Activatable membrane-interacting peptides and methods of use

ABSTRACT

The present disclosure provides activatable and detectable membrane-interacting peptides that, following activation, can interact with phospholipid bilayers, such as cell membranes. The present disclosure also provides methods of use of such compounds. The compounds of the present disclosure are of the general structure X 1a -A-X 2 -Z-X 1b , where A is a membrane-interacting peptide region having a plurality of nonpolar hydrophobic amino acid residues that, following separation from portions Z, is capable of interaction with a phospholipid bilayer; Z is an inhibitory peptide region that can inhibit the activity of portion A; X 2  is a cleavable linker that can be cleaved to release cleavage products from the compound; and X 1a  and X 1b  are optionally-present chemical handles that facilitate conjugation of various cargo moieties to the compound. Prior to cleavage of the composition at X 2 , the composition acts as a promolecule that does not associate with cellular membranes to a significant or detectable level. Following cleavage at cleavable linker X 2 , the cleavage product including portion A is free to interact with a phospholipid bilayer (e.g., a cell membrane), and thus accumulate at a site associated with a cleavage-promoting environment. Detection of the membrane-associated cleavage product can be accomplished by detection of a moiety attached through X 1a  and/or X 1b . Such compositions can be used in a variety of methods, including, for example, use in directly imaging active clotting within a subject.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority benefit of the filing date of U.S.Provisional Patent Application Ser. No. 61/785,450, filed on Mar. 14,2013, the disclosure of which application is herein incorporated byreference in its entirety.

INTRODUCTION

Detection and diagnosis of disease is generally an important first stepin selection of an appropriate therapy. However, many diseases andconditions involve biological processes that are difficult to detectwith sufficient sensitivity and specificity to enable a correctdiagnosis. Proteolytic events are an example of such a biologicalprocess and are directly or indirectly associated with a wide variety ofdiseases and conditions.

For example, despite gains in prevention and therapy, coronary heartdisease remains a leading cause of mortality. The event that causesdeath is most often a blood clot in the coronary artery initiated byrupture of an atherosclerotic plaque. Such clots can directly occludethis major vessel or break off and migrate to smaller arteries causingmyocardial ischemia and cellular damage. Current diagnostic toolsinclude assays that provide for indirect assessment of cardiac damageusing protein biomarkers. Such indirect assessment methods often aretime consuming, as they frequently require obtaining multiple samplesfrom a patient to monitor a change in biomarker levels over a period oftime. In addition, biomarker assays provide little or no information asto a location and/or size of an active blood clot. More directprocedures can be used to identify an area of and extent of cardiacdamage, but such direct procedures tend to be invasive, and are oftennot appropriate for all patients. Conventional imaging approachesavailable to facilitate assessment of cardiac damage are generally notused as a first line screening at least in part due to their relativelyhigh cost, low sensitivity, and toxicity associated with the amount ofradiation or imaging agent required to facilitate visualization. Suchlimitations apply to the diagnosis of other thrombotic problems eitherdirectly (i.e. heart attack and stroke) or indirectly (i.e., cancer anddiabetes).

There is a need for tools to facilitate diagnosis of conditionsassociated with proteolytic activity.

SUMMARY

The present disclosure generally provides activatable and detectablemembrane-interacting peptides that, following activation, can interactwith phospholipid bilayers, such as cell membranes. The presentdisclosure also provides methods of use of such compounds.

The compounds of the present disclosure are of the general structureX^(1a)-A-X²-Z-X^(1b), where A is a membrane-interacting peptide regionhaving a plurality of nonpolar hydrophobic amino acid residues that,following separation from portion Z, is capable of interacting with aphospholipid bilayer; Z is an inhibitory peptide region that can inhibitthe activity of portion A; X² is a cleavable linker that can be cleavedto release cleavage products from the compound; and X^(1a) and X^(1b)are optionally-present chemical handles that facilitate conjugation ofvarious moieties to the compound. Prior to cleavage of the compositionat X², the composition acts as a promolecule that does not associatewith phospholipid bilayers to a significant or detectable level.Following cleavage at cleavable linker X², the cleavage productincluding portion A is free to interact with a phospholipid bilayer(e.g., a cell membrane), and thus accumulate at a site associated with acleavage-promoting environment. Detection of the membrane-associatedcleavage product can be accomplished by detection of a moiety attachedthrough X^(1a) and/or X^(1b). Such compositions can be used in a varietyof methods, including, for example, use in directly imaging activeclotting within a subject.

In some embodiments, the present disclosure provides molecules thatinclude the structure, from N-terminal to C-terminal or C-terminal toN-terminal: X^(1a)-A-X²-Z-X^(1b). wherein X^(1a) and/or X^(1b) may bepresent or absent, and when present comprise a nucleophilic moiety; A isa membrane-interacting polypeptide portion that, when separated fromportion Z, comprises an alpha-helical structure capable of insertinginto a phospholipid bilayer; Z is a polypeptide that, when linked toportion A through portion X², is effective to inhibit interaction ofportion A with a phospholipid bilayer; and X² is a cleavable linker,wherein X² joins portion A to portion Z, and wherein X² can be cleavedunder physiological conditions. In some embodiments, portion A includesabout 5 to about 30 amino acid residues. In some embodiments, portion Aincludes the amino acid sequenceX^(a)X^(b)X^(c)X^(d)X^(e)X^(f)Y^(a)X^(g)X^(h)Y^(b)Y*X^(i)X^(j), whereX^(a), X^(b), X^(c), X^(d), X^(e), X^(f), X^(g), X^(h), X^(i), and X^(j)are hydrophobic amino acid residues, Y^(a) and Y^(b) are hydrophilicamino acid residues, and Y* is a charged amino acid residue. In someembodiments, portion A includes the amino acid sequence FVQWFSKFLGRIL(SEQ ID NO: 2), or a conservative amino acid substitution thereof. Insome embodiments, portion A includes the amino acid sequenceFVQWFSKFLGKLL (SEQ ID NO: 3), or a conservative amino acid substitutionthereof. In some embodiments, portion A includes the amino acid sequenceILGTILGLLKGL (SEQ ID NO: 4). In some embodiments, portion A includes theamino acid sequence of Japonicin-1 (SEQ ID NO: 5). In some embodiments,portion A includes the amino acid sequence FFWLSKIF (SEQ ID NO: 11). Insome embodiments, portion A includes fewer than 5 basic amino acidresidues.

In some embodiments, portion Z includes a covalently linked watersoluble polymer. In some embodiments, X² is enzymatically cleavable. Insome embodiments, X² is cleavable by thrombin. In some embodiments, X²is an enzymatically cleavable linker and Z includes an exositerecognition sequence for an enzyme that is capable of cleaving X². Insome embodiments, X² is cleavable by thrombin and the exositerecognition sequence is derived from the thrombin exosite recognitionsequence of a protease-activated receptor-1 (PAR-1) (SEQ ID NO: 18). Insome embodiments, X² is cleavable by TMPRSS2 and the exosite recognitionsequence is derived from the TMPRSS2 exosite recognition sequence of aprotease-activated receptor-2 (PAR-2) (SEQ ID NO: 20). In someembodiments, Z includes the amino acid sequence SFLL(X^(a))NPNDKYEPFW,wherein X^(a) is R or Q (SEQ ID NO: 23). In some embodiments, Z includesthe amino acid sequence KVDGTSHVTGDDD (SEQ ID NO: 20). In someembodiments, one or more of X^(1a), X^(1b), A, or Z includes a D-aminoacid. In some embodiments, X^(1a) is present and includes a nucleophilicmoiety. In some embodiments, X^(1b) is present and includes anucleophilic moiety. In some embodiments, the nucleophilic moiety of X^(1a) or X^(1b) includes a thiol functional group. In some embodiments,X^(1a) or X^(1b) includes an amino acid residue that includes thenucleophilic moiety. In some embodiments, the amino acid residue is acysteine residue. In some embodiments, the amino acid residue is alysine residue.

In some embodiments, X^(1a) or X^(1b) includes a cargo moiety covalentlyattached to the nucleophilic moiety. In some embodiments, the cargomoiety is a detectable moiety. In some embodiments, the detectablemoiety includes a fluorescent moiety. In some embodiments, thedetectable moiety comprises a radioisotope. In some embodiments, thepresent disclosure provides nucleic acids encoding the moleculedescribed above. In some embodiments, the present disclosure providescompositions that include the molecules described above and apharmaceutically acceptable carrier.

In some embodiments, the present disclosure provides methods ofdetectably labeling a cell, the methods including contacting a cell witha molecule as described above, wherein when the contacting is underconditions suitable for cleavage of the cleavable linker, the moleculeis cleaved to release the membrane interacting polypeptide portion forinteraction with a phospholipid bilayer of the cell and detectablylabels the cell. In some embodiments, the cell is in vivo. In someembodiments, the subject is a human.

In some embodiments, the present disclosure provides methods fordetection of a blood clot in a subject, the methods includingadministering to the subject a molecule as described above, wherein X²is cleavable by thrombin, wherein in the presence of thrombin themolecule is cleaved to release a cleavage product comprising thedetectable moiety and the membrane interacting polypeptide portion andwherein the cleavage product interacts with a phospholipid bilayer of acell in an area of thrombin enzyme activity, and detecting the presenceor absence of the detectable label of the cleavage product, wherein thepresence of the detectable label indicates an area of thrombin enzymeactivity associated with active clotting.

In some embodiments, Z comprises an amino acid sequence of an exositerecognition sequence for thrombin. In some embodiments, Z comprises anamino acid sequence of an exosite recognition sequence for TMPRSS2. Insome embodiments, Z comprises the exosite recognition sequence ofprotease-activated receptor-1 (PAR-1) (SEQ ID NO: 18). In someembodiments, Z comprises the exosite recognition sequence ofprotease-activated receptor-2 (PAR-2) (SEQ ID NO: 20).

In some embodiments, the present disclosure provides methods of making amolecule useful in delivery of a cargo moiety to a phospholipid bilayer,the methods including synthesizing the molecule as described above,wherein X^(1a) is present, and attaching a cargo moiety to thenucleophilic moiety of X^(1a), wherein a molecule useful in delivery ofa cargo moiety to a phospholipid bilayer is produced. In someembodiments, the synthesizing involves culturing a recombinant host cellcomprising an expression construct encoding the molecule. In someembodiments, the synthesizing is by chemical synthesis.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a schematic diagram of an example of a promolecule of thepresent disclosure. The promolecule is of the general structureX^(1a)-A-X²-Z. The membrane-interacting portion A can be conjugated toan imaging modality via portion X^(1a). Region A is linked to acleavable linker X², which is linked to a membrane-interactinginhibiting portion Z. After cleavage of X² by, e.g., an enzyme, portionA is separated from portion Z.

FIG. 2 is a schematic diagram that shows promolecules of the presentdisclosure undergoing a conformational change and inserting into cellmembranes. The schematic diagram shows that the process is dependentupon separation of portion Z from portion A following cleavage at X² by,e.g., a protease.

FIG. 3 shows the crystal structure of the protease thrombin bound to itsnatural substrate protease activated receptor-1 (PAR-1) (PDB ID 3LU9).The substrate (black sticks) has extensive interactions with the enzyme,and this sequence of amino acids is useful for targeting. At positionswhere the substrate has fewer interactions with the enzyme, and atpositions where the amino acid side chains of the substrate interactwith solvent (indicated by arrows), modifications to the amino acidsequence of the substrate can be made with little impact on targetingspecificity.

FIG. 4 is a graph showing the estimated number of interactions of theprotease thrombin with each of the amino acid residues in its substratePAR-1 (PDB ID 3LU9). Residues with more than five estimated interactionsare likely to play an important role in substrate binding. Residues withfewer than five estimated interactions, e.g., R46, N47, and N49(indicated in FIG. 3), may be altered to modulate the activity of thepromolecule without adversely impacting substrate binding.

FIG. 5 is a series of graphs showing the intrinsic fluorescence of asingle tryptophan residue in various promolecules of the presentdisclosure under different conditions. Panel A shows that prior toactivation, the spectral properties of the promolecule do not change inthe presence of liposomes composed of phosphatidylcholine (PC) orphosphatidylserine (PS) compared to buffer alone (PBS). Panel B showsthat the activated form of the promolecule presents a marked blue shiftin maximum wavelength, indicating that the cleavage product inserts intothese membranes.

FIG. 6 shows the differential behavior of a promolecule of the presentdisclosure as evidenced by förster resonance energy transfer (FRET).Panel A shows that prior to activation, the promolecules do notassociate closely enough with the liposomes to enable FRET from the1,1′-dioctadecyl-3,3,3′, 3′-tetra-methylindo-carbocyanine perchlorate(DiL). Panel B shows that the activated form exhibits a significant FRETsignal, indicating intimate association with DiL and the phospholipidmembrane.

FIG. 7 shows the differential behavior of a promolecule of the presentdisclosure as evidenced by circular dichroism spectroscopy. Panel Ashows that, when incubated with detergent micelles, the promoleculedisplays a weaker signature of alpha-helicity. Panel B shows that theactivated form displays a stronger signature of alpha-helicity.

FIG. 8 is a graph showing the intrinsic fluorescence of a promolecule asa function of time in the presence or absence of the enzyme thrombin.Incubation of the promolecule with thrombin (2 nM) leads to efficientactivation of the promolecule and a concomitant increase in interactionwith liposomes. In the absence of enzyme, there are no changes in thespectral signature of the promolecule, indicating an absence ofinteraction with the liposomes.

FIG. 9 shows the selective activation of a promolecule of the presentdisclosure as evidenced by the ability of Jurkat cells to exclude trypanblue dye. Cells (100,000) were incubated with the membrane-interactingpeptide-containing cleavage product (aAP1) or the promolecule form(proAP1) of the compound at concentrations of 1, 10 or 100 μM in 100 μLvolume for 2 hours, and were then assessed for trypan blue dye exclusionas a measurement of cell viability and membrane integrity. Highconcentrations of the membrane-interacting peptide-containing cleavageproduct led to cell death, while high concentrations of the promoleculedid not. Co-incubation of the promolecule (50 μM) with the proteasesthrombin (5 nM) or plasmin (200 nM) or coagulation factor Xa (200 nM)demonstrates that thrombin selectively activates the promolecule.

FIG. 10 shows exclusion of the fluorescent cell viability dye DRAQ7 bymouse pancreatic duct carcinoma cells under various conditions. DRAQ7 ismore sensitive to membrane disruption than trypan blue dye and providessecondary validation. Images were obtained with an epifluorescentmicroscope. (Panel A) Cells were stained with wheat germ agglutininconjugated to Oregon Green 488 to visualize their surfaces. (Panel B)Cells incubated with 100 μM of the membrane-interactingpeptide-containing cleavage product were sufficiently permeabilized toenable uptake of DRAQ7, which becomes fluorescent upon interaction withdeoxyribonucleic acid inside the cell. (Panel C) Cells were incubatedwith the promolecule for one hour. Without activation, the promoleculedoes not lead to significant uptake of DRAQ7 dye by cells. (Panel D)Cells were incubated with both a promolecule and thrombin for 30minutes. Addition of thrombin (10 nM) with incubation for 30 minutescauses activation of the promolecule, resulting in permeabilization ofthe membrane and uptake of DRAQ7 dye by the cells.

FIG. 11 shows exclusion of the fluorescent cell viability dye DRAQ7 bymouse X3 fibroblast cells under various conditions. Cells were incubatedwith a solution containing the promolecule (100 μM), or a solutioncontaining the membrane-interacting peptide-containing cleavage product(100 μM). Incubation with the promolecule did not lead to extensiveuptake of the DRAQ7 dye. Incubation with the membrane-interactingpeptide-containing cleavage product resulted in permeabilization of themembrane and uptake of DRAQ7 dye, which becomes fluorescent upon theinteraction with deoxyribonucleic acid inside the cell.

FIG. 12 shows exclusion of the fluorescent cell viability dye DRAQ7 bymouse X3 fibroblast cells under various conditions. The cell line wasgenetically altered to contain a variant of histone 2A conjugated togreen fluorescent protein (GFP), which enabled cell counting. Additionof thrombin (5 nM) and the promolecule (100 μM) caused the number ofpermeabilized cells to increase as a function of time. In the absence ofthrombin, permeabilization did not occur at a significant rate over thetime frame of the experiment.

FIG. 13 shows localization of the membrane-interactingpeptide-containing cleavage product to blood clots formed in vitro. Redblood cells appear as small, spherical cells and do not accumulate afluorescent signal, as they are not sites of clot formation.

FIG. 14 shows images of the lungs of a mouse injected with various dosesof thromboplastin to induce formation of emboli in the lungs. Panels Aand B show lungs from a mouse receiving a non-lethal dose ofthromboplastin. Panels C and D show lungs from a mouse receiving a fataldose of thromboplastin. Panel A shows a visual light image of the lungsof a mouse receiving a non-fatal dose, with areas of emboli formationvisible as white regions. Panel B shows a fluorescent microscope imageof the same lungs, with regions of membrane-interactingpeptide-containing cleavage product accumulation visible in areas ofemboli formation. Panel C shows a visual light image of the lungs of amouse receiving a fatal dose, with areas of emboli formation visible aswhite regions. Panel D shows a fluorescent microscope image of the samelungs, with regions of membrane-interacting peptide-containing cleavageproduct accumulation visible in areas of emboli formation.

FIG. 15 shows an image obtained using a small animal fluorescence andnear-infrared fluorescence imaging system. A puncture wound to the hindleg of an animal was visualized by detecting accumulation of themembrane-interacting peptide-containing cleavage product at the woundsite.

FIG. 16 is a graph showing the intensity of a fluorescent dye signal asa function of time. A puncture wound was inflicted to the hind leg of ananimal, and a region of interest was drawn at the site of wounding. Theintensity of the fluorescent signal coming from the region was plottedas a function of time. The resulting curve was used to quantify the rateof clot formation.

FIG. 17 shows images and data collected from an animal that wasadministered promolecules of the present disclosure. Panel A shows afluorescent microscope image taken before administration of thepromolecule. Panel B shows a fluorescent microscope image taken one hourafter administration of the promolecule. Panel C shows a fluorescentmicroscope image taken 24 hours after administration of the promolecule.Panel D shows a fluorescent microscope image of the duodenum of theanimal. Panel E shows a graph of signal intensity of the fluorescent dyeemanating from the bladder of the animal as a function of time.

FIG. 18 shows data obtained by reverse phase high pressure liquidchromatography (HPLC). Panel A shows data obtained from solutions of thepromolecule incubated with different proteases at the same concentration(2 nM) for 30 minutes at 37° C. Panel B shows a graph plotting theactivation kinetics of the promolecule when incubated with variousproteases.

FIG. 19 shows data obtained using fluorescence resonance energy transfer(FRET). Promolecules and membrane-interacting peptide-containingcleavage products where incubated with liposomes having a 100 nmdiameter and composed phosphatidylcholine and phosphatidylserine in a3:1 molar ratio (PC:PS) or entirely of phosphatidylserine (PS) and3,3′-dioctadecyloxacarbocyanine perchlorate (DiO). Panel A shows thatprior to activation, the promolecules do not associate closely enoughwith the PS liposomes to enable FRET from the DiO. In contrast, theactivated form exhibits a significant FRET signal indicating intimateassociation with DiO and phospholipid membranes containing PS. Panel Bshows that if the liposomes are formulated to have lessphosphatidylserine, the membrane-interacting peptide-containing cleavageproduct does not appear to interact with sufficient intimacy to enableFRET after activation by thrombin.

FIG. 20 shows the localization of membrane-interactingpeptide-containing cleavage products in blood clots formed in vitro. Redblood cells appear as small, spherical cells on the periphery of theslide and do not accumulate a fluorescent signal, as they are not sitesof clot formation. Panel A shows an image visualizing Cy3 fluorescentdye alone. Panel B shows a composite image of Cy3 fluorescent dye andbrightfield signals. Panel C is an image showing brightfield signalalone.

FIG. 21 shows a schematic representation of a promolecule of the presentdisclosure used for detection of proteolysis by a target enzyme. Uponactivation by an enzyme, a promolecule is converted into amembrane-interacting form, and accumulates in the vicinity of acleavage-promoting environment, enabling detection of a particulardisease or condition.

FIG. 22 shows data obtained from intrinsic fluorescence analysis. PanelA shows that, prior to activation, the spectral properties of thepromolecule undergo a limited change in the presence of liposomescomposed of phosphatidylcholine (PC) or phosphatidylserine (PS) comparedto buffer alone (PBS). Panel B shows that the membrane-interactingpeptide-containing cleavage product presents a marked blue shift inmaximum wavelength, indicating that it inserts into membranes.

FIG. 23 is a graph showing the uptake of trypan blue dye by MDA-MB-231cells under various conditions. High concentrations of themembrane-interacting peptide-containing cleavage product led to celldeath, while high concentrations of the promolecule did not.

FIG. 24 is a graph showing the uptake of trypan blue dye by MDA-MB-231cells under various conditions. Panel A shows that a promolecule cleavedby thrombin is selectively activated by thrombin. Panel B shows that apromolecule cleaved by matriptase is selectively activated bymatriptase.

FIG. 25 shows microscopic images of HT29 cells incubated withpromolecules of the present disclosure. The surfaces of HT29 cells werelabeled with wheat germ agglutinin conjugated to Oregon Green 488. Afterco-incubation of a promolecule (10 nM) with HT29 cells for 20 minutes, afluorescent signal from the promolecule was detectable on the surface ofcells (Panel A). Subsequent washing and imaging 24 hours later revealedthat the fluorescent signal from the promolecule was localized inpunctate spheres inside the cells (Panel B).

Before the present invention is further described, it is to beunderstood that this invention is not limited to particular embodimentsdescribed, as such may, of course, vary. It is also to be understoodthat the terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to be limiting, sincethe scope of the present invention will be limited only by the appendedclaims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range, is encompassed within the invention. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges, and are also encompassed within the invention, subjectto any specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either orboth of those included limits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present invention, the preferredmethods and materials are now described. All publications mentionedherein are incorporated herein by reference to disclose and describe themethods and/or materials in connection with which the publications arecited.

It must be noted that as used herein and in the appended claims, thesingular forms “a,” “an,” and “the” include plural referents unless thecontext clearly dictates otherwise. Thus, for example, reference to “themolecule” includes reference to one or more proteins, and so forth. Itis further noted that the claims may be drafted to exclude any optionalelement. As such, this statement is intended to serve as antecedentbasis for use of such exclusive terminology as “solely,” “only” and thelike in connection with the recitation of claim elements, or use of a“negative” limitation.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention.Further, the dates of publication provided may be different from theactual publication dates which may need to be independently confirmed.

DEFINITIONS

The terms “polypeptide,” “oligopeptide,” “peptide,” and “protein,” usedinterchangeably herein, refer to a polymeric form of amino acids of anylength, which can include genetically coded and non-genetically codedamino acids, chemically or biochemically modified or derivatized aminoacids, and polypeptides having modified peptide backbones. The termincludes fusion proteins, including, but not limited to, fusion proteinswith a heterologous amino acid sequence, fusion proteins withheterologous and homologous leader sequences, with or without N-terminalmethionine residues; immunologically tagged proteins; and the like.

The term “membrane-interacting peptide” refers to a peptide moleculehaving a plurality of nonpolar hydrophobic amino acid residues, and,when unconstrained by a portion Z as described herein, comprises analpha-helical structure capable of interaction with phospholipidbilayers such as a cell membrane. Such secondary structure may appearbefore, during or after insertion of the membrane-interacting peptideinto the phospholipid bilayer. The composition of membrane-interactingpeptides as described herein is not strictly limited to nonpolarhydrophobic amino acid residues, as such peptides may include differenttypes of amino acid residues, for example, polar uncharged, polar basic,or polar acidic amino acid residues as well.

The term “antimicrobial polypeptide” refers to a type ofmembrane-interacting peptide that is derived from a naturally-occurringpeptide that exhibits antimicrobial activity in its natural form basedon its ability to interact with cell membranes. It is understood thatthe term “antimicrobial polypeptide” as used herein does not require orimply that the polypeptides so described have antimicrobial activity.Any peptide shown to spontaneously interact with and potentially insertinto phospholipid membranes are included in this category. For example,spontaneously inserting membrane interaction peptides from naturallyoccurring transmembrane proteins may be applied. Antimicrobialpolypeptides are well known in the art, and include, for example,polypeptides in the temporin family of proteins.

The term “promolecule” as used herein refers to a molecule whoseactivity is restricted because the individual portions of the moleculeare linked together, therefore limiting or restricting the activity thatthe individual portions may have when not linked to one another. Theactivity of the individual portions of a promolecule is unleashed uponcleavage or disruption of the bonds that hold the individual portionstogether.

The term “enzyme-activated” refers to a molecule whose behavior ismodified by an enzyme. Many activating enzymes fall under the class ofhydrolases EC 3.1 to EC 3.13 or peptidases EC 3.4 to 3.99 in theNomenclature Committee of the International Union of Biochemistry andMolecular Biology (NC-IUBMB). Example enzyme activities include thosethat act upon bonds of the type ether, peptide, carbon-nitrogen, acidanhydrides, carbon-carbon, halide, phosphorus-nitrogen, sulfur-nitrogen,carbon-phosphorus, sulfur-sulfur, carbon-sulfur.

The term “non-standard amino acid” means any molecule other than anaturally-occurring amino acid molecule that can be incorporated into apeptide backbone of a polypeptide in lieu of a naturally-occurring aminoacid residue in a polypeptide. Non-limiting examples of suchnon-standard amino acids include: hydroxylysine, desmosine,isodesmosine, or others.

The term “modified amino acid” means any naturally-occurring amino acidthat has undergone a chemical or biochemical modification, such as apost-translational modification. Non-limiting examples of modified aminoacids include: methylated amino acids, (e.g. methyl histidine,methylated lysine) acetylated amino acids, amidated amino acids,formylated amino acids, hydroxylated amino acids, phosphorylated aminoacids, or others.

As used herein, “homologues” or “variants” refers to protein sequencesthat are similar based on their amino acid sequences. Homologues andvariants include proteins that differ from naturally-occurring sequencesby one or more conservative amino acid substitutions.

As used herein, the term “conservative amino acid substitution” means asubstitution of an amino acid residue for another amino acid residuehaving similar chemical properties.

The term “treatment” as used herein means that at least an ameliorationof the symptoms associated with a disease or condition afflicting thesubject is achieved, where amelioration refers to at least a reductionin the magnitude of a parameter, e.g., a symptom, associated with thedisease or condition being treated. As such, treatment includessituations where the condition, or at least symptoms associatedtherewith, are reduced or avoided.

It will be appreciated that throughout the present disclosure referenceis made to amino acids according to the single letter or three lettercodes. For the reader's convenience, the single and three letter aminoacid codes are provided below. In addition, the amino acid residuesprovided below are divided into categories based on their chemicalproperties. The headings provided in the table below (Nonpolar,Hydrophobic; Polar, Uncharged; Polar, Acidic; and Polar, Basic) are usedto refer generally to amino acid residues having the identified chemicalproperties.

Nonpolar, Hydrophobic Residues Alanine Ala A Valine Val V Leucine Leu LIsoleucine Ile I Phenylalanine Phe F Tryptophan Trp W Methionine Met MProline Pro P Polar, Acidic Aspartic Acid Asp D Glutamic Acid Glu E

Polar, Uncharged Residues Glycine Gly G Serine Ser S Threonine Thr TCysteine Cys C Tyrosine Tyr Y Asparagine Asn N Glutamine Gln Q Polar,Basic Lysine Lys K Arginine Arg R Histidine His H

The terms “nucleic acid molecule” and “polynucleotide” are usedinterchangeably and refer to a polymeric form of nucleotides of anylength, either deoxyribonucleotides or ribonucleotides, or analogsthereof. Non-limiting examples of polynucleotides include linear andcircular nucleic acids, messenger RNA (mRNA), cDNA, recombinantpolynucleotides, vectors, probes, and primers.

The term “heterologous” refers to two components that are defined bystructures that can be derived from different sources. For example,where “heterologous” is used in the context of a polypeptide, thepolypeptide includes operably linked amino acid sequences that can bederived from polypeptides having different amino acid sequences (e.g., afirst amino acid sequence from a first polypeptide and a second aminoacid sequence from a second polypeptide). Similarly, “heterologous” inthe context of a polynucleotide encoding a chimeric polypeptide includesoperably linked nucleic acid sequences that can be derived fromdifferent genes (e.g., a first component from a nucleic acid encoding afirst portion of a peptide according to an embodiment disclosed hereinand a second component from a nucleic acid encoding a second portion ofa peptide disclosed herein).

“Derived from” in the context of an amino acid sequence orpolynucleotide sequence (e.g., a polypeptide derived from anantimicrobial peptide) is meant to indicate that the polypeptide ornucleic acid has a sequence that is based on that of a referencepolypeptide or nucleic acid, and is not meant to be limiting as to thesource or method in which the protein or nucleic acid is made.

The term “operably linked” refers to functional linkage betweenmolecules to provide a desired function. For example, “operably linked”in the context of a polypeptide refers to a functional linkage betweenamino acid sequences (e.g., of different domains) to provide for adescribed activity of the polypeptide. “Operably linked” in the contextof nucleic acids refers to a functional linkage between nucleic acids toprovide a desired function such as transcription, translation, and thelike, e.g., a functional linkage between a nucleic acid expressioncontrol sequence (such as a promoter, signal sequence, or array oftranscription factor binding sites) and a second polynucleotide, whereinthe expression control sequence affects transcription and/or translationof the second polynucleotide.

As used herein in the context of the structure of a polypeptide,“N-terminus” and “C-terminus” refer to the extreme amino and carboxylends of the polypeptide, respectively, while “N-terminal” and“C-terminal” refer to relative positions in the amino acid sequence ofthe polypeptide toward the N-terminus and the C-terminus, respectively,and can include the residues at the N-terminus and C-terminus,respectively. “Immediately N-terminal” or “immediately C-terminal”refers to a position of a first amino acid residue relative to a secondamino acid residue where the first and second amino acid residues arecovalently bound to provide a contiguous amino acid sequence.

“Isolated” refers to a protein of interest (e.g., a membrane-interactingpeptide) that, if naturally occurring, is in an environment differentfrom that in which it may naturally occur. “Isolated” is meant toinclude proteins that are within samples that are substantially enrichedfor the protein of interest and/or in which the protein of interest ispartially or substantially purified. Where the protein is not naturallyoccurring, “isolated” indicates the protein has been separated from anenvironment in which it was made by either synthetic or recombinantmeans.

“Enriched” means that a sample is non-naturally manipulated (e.g., by anexperimentalist or a clinician) so that a protein of interest is presentin a greater concentration than the concentration of the protein in thestarting sample, such as a biological sample (e.g., a sample in whichthe protein naturally occurs or in which it is present afteradministration), or in which the protein was made (e.g., as in abacterial protein and the like).

“Substantially pure” indicates that an entity makes up greater thanabout 50% of the total content of the composition (e.g., total proteinof the composition), or greater than about 60% of the total proteincontent. For example, a “substantially pure” peptide refers tocompositions in which at least 75%, at least 85%, at least 90% or moreof the total composition is the entity of interest (e.g. 95%, 98%, 99%,greater than 99%), of the total protein. The protein can make up greaterthan about 90%, or greater than about 95% of the total protein in thecomposition.

The term “binding” refers to a direct association between two molecules,due to, for example, covalent, electrostatic, hydrophobic, and ionicand/or hydrogen-bond interactions, including interactions such as saltbridges and water bridges.

The term “nucleophilic moiety” as used herein refers to a functionalgroup, which comprises a nucleophilic reactive group. A nucleophilicreactive group comprises at least one pair of free electrons that isable to react with an electrophile. Examples of nucleophilic moietiesinclude sulfur nucleophiles, such as thiols, thiolate anions, anions ofthiolcarboxylate, anions of dithiocarbonates, and anions ofdithiocarbamates; oxygen nucleophiles, such as hydroxide anion,alcohols, alkoxide anions, and carboxylate anions; nitrogennucleophiles, such as amines, azides, and nitrates; and carbonnucleophiles, such as alkyl metal halides and enols.

The terms “patient” or “subject” as used interchangeably herein canrefer to a human or to a non-human animal, e.g. a mammal, includinghumans, primates, domestic and farm animals, and zoo, sport, laboratory,or pet animals, such as horses, cows, dogs, cats, rodents, and the like.

DETAILED DESCRIPTION

Overview

The present disclosure generally provides activatable and detectablemembrane-interacting peptides that can be used to identify areas of asubject that are associated with a particular biological activity, e.g.,proteolysis. Following activation, the promolecules of the presentdisclosure are capable of forming alpha-helical structures that interactwith and insert into phospholipid bilayers, such as cell membranes. Thepresent disclosure also provides methods of use of such compounds.

The compounds of the present disclosure find use in, for example,methods relating to the diagnosis of disease. For example, anactivatable membrane-interacting peptide having a portion X² that iscleavable by the enzyme thrombin can be administered to a subjectsuspected of having a condition associated with thrombin activity, suchas active blood clotting. In this example, exposure of the molecule toan area of thrombin activity in the subject results in cleavage at X² togenerate a cleavage product containing portion A, which cleavage productis capable of inserting into cell membranes in the area of thrombinactivity and thus in the area of active clotting. Detection of thiscleavage product in cell membranes can be accomplished by imaging of thetissue(s) suspected of being associated with active clotting to image adetectable moiety attached to portion A through portion X^(1a). Thepresence of thrombin activity in a subject can also be assessed bydetection of the cleavage product containing portion Z, which may befacilitated by moieties attached as portion X^(1b).

The compositions of the present disclosure can be used in a variety ofmethods, including, e.g., use in directly imaging active clotting,infection, or malignancy within a subject.

Activatable Membrane-Interacting Peptides

The promolecules of the present disclosure are of the general structure,from N-terminus to C-terminus or from C-terminus to N-terminus:A-X²-Zwhere

A is a membrane-interacting peptide region having a plurality ofnonpolar hydrophobic amino acid residues that, following cleavage fromthe composition, comprises an alpha-helical structure capable ofinteracting with a phospholipid bilayer (FIG. 1);

Z is an inhibitory peptide region that can inhibit the activity ofportion A and, in some embodiments, can facilitate targeted interactionof a promolecule with a specific enzyme; and

X² is a cleavable linker that can be cleaved to release cleavageproducts from the compound.

Prior to cleavage of the composition at X², the composition acts as apromolecule that does not significantly or detectably associate withphospholipid bilayers. Cleavage of X² results in the formation of acleavage product comprising portion A and a cleavage product comprisingportion Z. Following cleavage of X², the cleavage product comprisingportion A, now unconstrained by portion Z, is free to interact with aphospholipid bilayer (e.g., a cell membrane), and thus accumulate at asite associated with a cleavage-promoting environment (FIG. 2).

In some embodiments, the promolecules of the present disclosure are ofthe general structure, from N-terminus to C-terminus or from C-terminusto N-terminus:X^(1a)-A-X²-Z-X^(1b)where

A, X², and Z are as described above; and

X^(1a) and X^(1b) are optionally-present chemical handles thatfacilitate conjugation of various moieties to the compound.

Detection of cleavage products comprising portion A or portion Z can beaccomplished by detection of a detectable moiety attached throughchemical handle X^(1a) or X^(1b), or by other methods, e.g., detectionusing an antibody that specifically binds to an amino acid sequence ofthe cleavage product.

The various features of the compounds and methods of the presentdisclosure are described in more detail below.

The overall length of the intact structure X^(1a)-A-X²-Z-X^(1b) may varybased on the sizes of the individual portions that are used to assemblea given molecule. In some embodiments, the overall size of the intactstructure is up to about 15 amino acids in length. In some embodiments,the overall length of the intact structure is up to about 20, up toabout 30, up to about 40, up to about 50, up to about 60, up to about70, up to about 80, up to about 90, up to about 100, or up to about 110amino acids in length. In some embodiments, the overall length of theintact structure may be from about 15 to about 20, about 20 to about 30,about 30 to about 40, about 40 to about 50, about 50 to about 60, about60 to about 70, about 70 to about 80, about 80 to about 90, about 90 toabout 100, or about 100 to about 110 amino acids in length. The overalllength of the intact structure is no more than about 115 amino acids inlength.

The intact structure X^(1a)-A-X²-Z-X^(1b) may be referred to herein as a“promolecule.” Portion A of the promolecule does not significantlyinteract with phospholipid bilayers due to the presence of portion Z inthe promolecule. Without being held to theory, portion Z inhibits thephospholipid bilayer interacting properties of portion A by preventingportion A from forming an alpha-helical structure when portion A andportion Z are linked together by portion X². Following cleavage of X²,portion Z is separated from portion A, allowing the cleavage productcomprising portion A to undergo a conformational change such that atleast portion A can form a regular structure such as that of analpha-helical structure. In the alpha-helical conformation, portion Aspontaneously interacts with phospholipid bilayers, e.g., by insertinginto the phospholipid bilayer.

One of ordinary skill in the art will appreciate that the promoleculesof the present disclosure can be adapted for use in a variety ofsettings, e.g., by providing for cleavable linkers that differ inconditions that provide for cleavage. In some embodiments, a promoleculehas the structure, from N-terminus to C-terminus or from C-terminus toN-terminus, A-X²-Z. In some embodiments, a promolecule has thestructure, from N-terminus to C-terminus or from C-terminus toN-terminus, X^(1a)-A-X²-Z. In some embodiments, a promolecule has thestructure, from N-terminus to C-terminus or from C-terminus toN-terminus, A-X²-Z-X^(1b). In some embodiments, a promolecule has thestructure, from N-terminus to C-terminus or from C-terminus toN-terminus, X^(1a)-A-X²-Z-X^(1b). As disclosed herein, the variousembodiments of portions X^(1a), A, X², Z, and X^(1b) may be freelyinterchanged to form a molecule having any of the above-describedfeatures. In some embodiments, multiple copies of X^(1a) or X^(1b) maybe incorporated to enhance detection sensitivity or pharmacologicalproperties.

Promolecules of the present disclosure may be optionally modified togenerally provide, e.g., a longer circulating half-life, focusedactivity within the cardiovascular system, protection from non-specificdegradation, and/or greater sensitivity to certain imaging modalities.Such optional modifications include, e.g., conjugation of two or morepromolecules to a central polyethylene glycol molecule to a form adendrimer or amidation of the N- and/or C-termini of the promolecule tomitigate unwanted proteolysis.

Properties of Activatable Membrane-Interacting Peptides

As described above, the promolecules of the present disclosure have thegeneral structure X^(1a)-A-X²-Z-X^(1b). In order to prevent themembrane-interacting portion (portion A) from interacting with cellmembranes prior to activation, the promolecules are designed to have anisoelectric point (pI) of 7 or lower. The isoelectric point is the pH atwhich the net charge on a peptide molecule is zero. The pI of thefull-length promolecules of the present disclosure can be modulated byadjusting the pI of one or more of the individual portions X^(1a), A,X², Z, or X^(1b) that make up the promolecule, or by chemicallymodifying any or all portions of the compound (e.g., by phosphorylationor sulfation to impart additional negative charge).

The pI of a peptide can be modulated by substituting, eliminating, orintroducing amino acid residues in order to change the overall netcharge of the peptide. Decreasing the net charge of a peptide reducesits pI value. For example, eliminating one or more positively chargedamino acid residues (e.g. K, R, or H) or replacing such residues withuncharged or negatively charged residues reduces the pI value of thepeptide.

The pI of a given peptide can be readily determined by using a computeralgorithm for pI estimation (e.g., Protein Calculator, ScrippsInstitute). Such computer algorithms are readily available to the publicvia the internet and can determine a theoretical pI value for a peptidebased on its amino acid sequence. Promolecules of the present disclosureare designed to have a theoretical pI value less than or equal to 7.

The promolecules of the present disclosure are generally designed tohave an overall net charge preferably less than or equal to about zero.This can be accomplished, for example, by substituting, eliminating, orintroducing various amino acid residues in the polypeptide sequences ofportion A or portion Z, or by introducing charged moieties to portion Z,to neutralize the overall charge of the promolecule. Charged amino acidresidues or moieties that are introduced to neutralize the charge ofother amino acid residues or chemical moieties are placed as close aspossible to one another in order to maximize charge-cancelling effects,e.g., a distance of no more than about 40 Angstroms. In someembodiments, promolecules of the present disclosure need not have anoverall charge of less than or equal to about zero if, for example, thepropensity of the membrane interaction segment to spontaneously insertinto phospholipid membranes is limited.

Cargo moieties that are optionally conjugated to the promolecules of thepresent disclosure may be charged, and therefore may impact the pI valueand the overall net charge of a promolecule, thus impacting therestriction in membrane-interacting activity. The charge on a particularcargo moiety added to a promolecule will generally cancel or neutralizethe overall net charge of the promolecule that it is conjugated to andreduce the overall pI to a value of seven or lower. For example, apromolecule having an overall net charge of +1 could be conjugated to adetectable moiety having a charge of −1 to produce a molecule having anoverall net charge of zero. Water soluble fluorescent dyes, such asthose in the cyanine dye family, including Cy3, Cy5, and Cy7, areparticularly useful in this regard due to their zwitterionic nature fromtwo negatively-charged sulphate groups and a tertiary amine group. Metalchelating moieties, such as1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) ordiethylene triamine pentaacetic acid (DTPA) capable of bindingradioisotopes Gallium-68 or Technetium-99m are zwitterionic as well,bearing multiple positively- and negatively-charged moieties. Metalbinding to DOTA or DTPA occurs through the amine groups, thus allowingthese entities to impart a charge of up to −4.

Portion Z's ability to inhibit or prevent portion A from interactingwith phospholipid bilayers can also be modulated by changing the overalllength of portion Z. This can be done, for example, by adding amino acidresidues to portion Z, or by conjugating a molecule, such as awater-soluble polymer, to portion Z. In some embodiments, negativelycharged amino acids or similar chemical modifications, such asphosphates or sulphate moieties, are added to portion Z. In someembodiments, polyethylene glycol is conjugated to portion Z in order toincrease the length of portion Z and enhance its ability to inhibitportion A from interacting with cell membranes prior to activation. Insome embodiments, whole proteins (e.g. albumin) may be conjugated toportion Z. In certain embodiments, a polymer or protein that isconjugated to portion Z may also increase the circulating half-life ofthe promolecule. In some embodiments, the promolecules of the presentdisclosure may be conjugated to polymers having branched, dendrimeric,or otherwise polyvalent architecture.

Portion A—Membrane-Interacting Peptides

The membrane-interacting peptides of the present disclosure compriseamino acid sequences that are capable of forming alpha-helicalstructures, e.g., upon contacting an environment with a lower dielectricconstant than water. Following cleavage of X², the cleavage productcomprising portion A comprises an alpha-helical structure that iscapable of inserting into a phospholipid bilayer in the vicinity of thecleavage-promoting environment. Without being held to theory, thealpha-helical structure of portion A may be present in the moleculeprior to cleavage but, due to constraint by portion Z, is unable toinsert into a phospholipid bilayer. The presence of portion A and/orportion Z constrains portion A such that portion A does not form analpha-helical structure sufficient to allow for significant ordetectable insertion into a phospholipid bilayer.

An alpha helix is a common motif in the secondary structure of proteins,and generally comprises a right-handed coiled or spiral conformationthat is stabilized by hydrogen bonds in which the N—H group of a firstamino acid residue forms a hydrogen bond with the C═O group of an aminoacid residue located four residues away in the polypeptide chain. Atypical alpha helix comprises approximately 3.6 amino acid residues perturn of the helix, and is a tightly-packed structure. The side chains ofthe amino acid residues that make up an alpha helix face the outside ofthe helix. Different amino acid sequences have different propensitiesfor forming alpha helices due, in part, to the differing chemicalproperties of the amino acid side chains.

As described above, promolecules of the present disclosure generallycomprise a membrane-interacting peptide portion A. Portion A may bederived from a naturally-occurring polypeptide, or may be a variant of anaturally-occurring polypeptide. The overall length of portion A can be,for example, about 5 up to about 10 amino acids, or can be up to about15, up to about 20, up to about 25, or up to about 30 amino acids.Portion A may range in size from about 5 to about 10 amino acids inlength, or may be about 10 to about 15, about 15 to about 20, about 20to about 25, or about 25 to about 30 amino acids in length. Portion A isno longer than about 35 amino acid residues in length.

Membrane-interacting peptides generally comprise a plurality ofnonpolar, hydrophobic amino acid residues (e.g., alanines, valines,leucines, isoleucines, phenylalanines, tryptophans, methionines, orprolines), but may comprise other types of amino acids as well, such aspolar uncharged, polar acidic, and/or polar basic amino acid residues.In general, the membrane-interacting peptides of the present disclosurecomprise fewer than 5 polar basic amino acid residues. In someembodiments, the amino acid sequence of portion A comprises multipleregions of two to three contiguous nonpolar hydrophobic amino acidresidues interspersed with regions of one to two contiguous polaruncharged, polar acidic, or polar basic residues. After cleavage of X²,portion A undergoes a conformational change, typically forming analpha-helical structure that readily interacts with cell membranes(e.g., membranes present in the cells of eukaryotic, prokaryotic orarchael organisms, or artificial membranes of detergent micelles orliposomes of varying compositions, including synthetic polymers).

Antimicrobial Peptides

Antimicrobial peptides that elicit their effects through membraneinteraction are well known in the art, and include examples such as thetemporin family of proteins, which can be naturally obtained from theskin of frogs belonging to the Rana temporaria species. Antimicrobialpeptides generally comprise fewer than about 30 amino acid residues and,under physiological conditions, contain alpha-helical structures havingnonpolar hydrophobic amino acid residues that facilitate theirinteraction with the phospholipid bilayers of cell membranes. Suchinteractions may generally include types ranging from structuredbarrel-stave pores to broadly-defined detergent-like behavior. In someembodiments of the present disclosure, an amino acid sequence of anaturally-occurring antimicrobial peptide is utilized as amembrane-interacting peptide. In other embodiments, amembrane-interacting peptide that comprises modifications relative to anaturally-occurring antimicrobial peptide, e.g., elimination,introduction, or substitution of one or more amino acid residues,addition of chemical modifications such as disulfide bonds, or otherchemical modifications (e.g., amidation), is utilized as amembrane-interacting peptide. In other embodiments, the peptide sequenceis capable of spontaneous membrane interaction and/or insertion, but isnot associated with membrane disrupting activity.

Examples of antimicrobial peptides are provided below.

Modifications to Membrane-Interacting Peptides

Antimicrobial peptides or portions thereof may be incorporated into thecompounds of the present disclosure in their naturally-occurring form,or may be modified to alter their chemical properties and adapt such fora desired use. For example, the membrane-interaction potential ofantimicrobial peptides may be strengthened or weakened by, e.g., adding,eliminating or substituting certain amino acid residues in the proteinsequence. Such additions, eliminations, or substitutions can be made,e.g., to introduce charged amino acid residues, to eliminate chargedamino acid residues, to introduce hydrophobic amino acid residues, toeliminate hydrophobic amino acid residues, etc.

In some embodiments, an antimicrobial peptide sequence may be altered bychemically modifying the peptide with disulfide bonds or other chemicalmodifications (e.g. amidation). Many antimicrobial peptides arenaturally produced with such modifications to improve the potency oftheir interactions with phospholipid membranes and resistance toproteolysis.

Temporins

In some embodiments, portion A comprises a protein from the Temporinfamily. Proteins in the Temporin family generally range from about 10 upto about 14 amino acids in length. The consensus sequence for theTemporin family of proteins showing the most abundant amino acid foundat each position is: FLP(I/L)IASLL(S/G)KLL (SEQ ID NO: 1). The consensussequence for the Temporin family of proteins showing the general aminoacid type found at each position is:X^(a)X^(b)X^(c)X^(d)X^(e)X^(f)Y^(a)X^(g)X^(h)Y^(b)Y*X^(i)X^(j), whereX^(a), X^(b), X^(c), X^(d), X^(e), X^(f), X^(g), X^(h), X^(i), and X^(j)are hydrophobic amino acid residues, Y^(a) and Y^(b) are hydrophilicamino acid residues, and Y* is a charged amino acid residue. The tablebelow shows the amino acid sequences of several Temporin andTemporin-like peptides that are useful in the promolecules and methodsof the present disclosure.

As described above, antimicrobial peptide sequences may be altered byeliminating or substituting one or more of the amino acid residues. Forexample, in some embodiments, a membrane-interacting peptide comprisesTemporin-L, whose amino acid sequence is FVQWFSKFLGRIL (SEQ ID NO: 2).In other embodiments, a membrane-interacting peptide comprises aderivative of Temporin-L having the amino acid sequence FVQWFSKFLGKLL(SEQ ID NO: 3), wherein amino acid residues R and I at positions 11 and12 of the Temporin-L sequence have been replaced with amino acidresidues K and L, respectively.

TABLE 1 Amino acid sequences of Temporin and Temporin-like peptidesthirteen amino acids in length. Longer and shorter members of the familyhave also been described but are not included in this table. PeptideName Amino Acid Sequence Temporin-A — F L P L I G R V L S G I L — (SEQID NO: 61) Temporin-B — L L P I V G N L L K S L L — (SEQ ID NO: 62)Temporin-C — L L P I L G N L L N G L L — (SEQ ID NO: 63) Temporin-D — LL P I V G N L L N S L L — (SEQ ID NO: 64) Temporin-E — V L P I I G N L LN S L L — (SEQ ID NO: 65) Temporin-F — F L P L I G K V L S G I L — (SEQID NO: 66) Temporin-G — F F P V I G R I L N G I L — (SEQ ID NO: 67)Temporin-H — L S P — — — N L L K S L L — (SEQ ID NO: 68) Temporin-K — LL P — — — N L L K S L L — (SEQ ID NO: 69) Temporin-L — F V Q W F S K F LG R I L — (SEQ ID NO: 70) Temporin-1Ca — F L P F L A K I L T G V L —(SEQ ID NO: 71) Temporin-1Cb — F L P L F A S L I G K L L — (SEQ ID NO:72) Temporin-1Cc — F L P F L A S L L T K V L — (SEQ ID NO: 73)Temporin-1Cd — F L P F L A S L L S K V L — (SEQ ID NO: 74) Temporin-1Ce— F L P F L A T L L S K V L — (SEQ ID NO: 75) Temporin-1Ga S I L P T I VS F L S K V F — (SEQ ID NO: 76) Temporin-1Gb S I L P T I V S F L S K F L— (SEQ ID NO: 77) Temporin-1Gc S I L P T I V S F L T K F L — (SEQ ID NO:78) Temporin-1Gd F I L P L I A S F L S K F L — (SEQ ID NO: 79)Temporin-1La — V L P L I S M A L G K L L — (SEQ ID NO: 80) Temporin-1LbN F L G T L I N L A K K I M — (SEQ ID NO: 81) Temporin-1Lc — F L P I L IN L I H K G L L (SEQ ID NO: 82) Temporin-1P — F L P I V G K L L S G L L— (SEQ ID NO: 83) Ranatuerin-5 — F L P I — A S L L G K Y L — (SEQ ID NO:84) Ranatuerin-6 — F I S A I A S M L G K F L — (SEQ ID NO: 85)Ranatuerin-7 — F L S A I A S M L G K F L — (SEQ ID NO: 86) Ranatuerin-8— F I S A I A S F L G K F L — (SEQ ID NO: 87) Ranatuerin-9 F L F P L I TS F L S K V L — (SEQ ID NO: 88) Peptide A1 — F L P A I A G I L S Q L F —(SEQ ID NO: 89) Peptide B9 — F L P L I A G L L G K L F — (SEQ ID NO: 90)

In some embodiments of the present disclosure, a membrane-interactingpeptide comprises a Temporin or a Temporin-like peptide listed in Table1, or a conservative amino acid substitution thereof. In someembodiments of the present disclosure, a membrane-interacting peptidecomprises the sequence of Temporin-L (FVQWFSKFLGRIL) (SEQ ID NO: 2), ora conservative amino acid substitution thereof.

Protonectin

In some embodiments of the present disclosure, a membrane-interactingpeptide comprises Protonectin, having the amino acid sequenceILGTILGLLKGL (SEQ ID NO: 4), or a conservative amino acid substitutionthereof.

Japonicins

In some embodiments, a membrane-interacting peptide may comprise aJaponicin or a Japonicin-like peptide listed in Table 2, or aconservative amino acid substitution thereof. In some embodiments of thepresent disclosure, a membrane-interacting peptide comprises thesequence of Japonicin-1 (FFPIGVFCKIFKTC) (SEQ ID NO: 5), or aconservative amino acid substitution thereof. Japonicins are naturallyobtainable from the skin of the Japanese brown frog Rana japonica andrange in length from about 14 up to about 21 amino acid residues. Thetable below shows the amino acid sequences of several Japonicin andJaponicin-like peptides that are useful in the promolecules and methodsof the present disclosure.

TABLE 2 Amino acid sequences of Japonicin and Japonicin-like peptides.Peptide Name Amino Acid Sequence Japonicin-1 F — — — — — — F P I G V F CK I F K — T C (SEQ ID NO: 5) Japonicin- F — — — — — — F P L A L L C K VF K — K C 1CDYa (SEQ ID NO: 92) Japonicin- F — — — — — — L L F P L M C KI Q G — K C 1Npa (SEQ ID NO: 93) Japonicin- F — — — — — — V L P L V M CK I L R — K C 1Npb (SEQ ID NO: 94) Japonicin-2 F G L P M L S I L P K A LC I L L K R K C (SEQ ID NO: 95)

In some embodiments of the present disclosure, a membrane-interactingpeptide comprises a Japonicin or a Japonicin-like peptide listed inTable 2, or a conservative amino acid substitution thereof.

Additional Peptides

In addition to the peptides described above, promolecules of the presentdisclosure may comprise a membrane-interacting peptide listed in thefollowing table, or a conservative amino acid substitution thereof. Insome embodiments, the peptides listed in the table below comprise N-and/or C-terminal modifications that may modulate their activity.

TABLE 3 Peptides suitable for use in portion A. Amino Acid Peptide NameSequence SEQ ID NO: Combi-1 RRWWRF SEQ ID NO: 6 Combi-2 FRWWHR SEQ IDNO: 7 Jelleine-1 PFKLSLHL SEQ ID NO: 8 Jelleine-2 TPFKLSLHL SEQ ID NO: 9Temporin-SHf FFFLSRIF SEQ ID NO: 10 Modified Temporin-SHF FFWLSKIF SEQID NO: 11 Jcpep7 KVFLGLK SEQ ID NO: 12 Myxinidin GIHDILKYGKPS SEQ ID NO:13 1T51 ILGKIWEGIKSLF SEQ ID NO: 14 Mastoparan B LKLKSIVSWAKKVL SEQ IDNO: 15 K4 KKKKPLFGLFFGLF SEQ ID NO: 16 Agelaia-MP INWLKLGKAIIDAL SEQ IDNO: 17

Portion Z—Membrane-Interaction Inhibitory Peptide

Compounds of the present disclosure generally comprise portion Z, whichinhibits or prevents portion A from interacting with phospholipidbilayers when linked to portion A through portion X². In someembodiments, portion Z also facilitates the interaction of thepromolecule with a target enzyme.

Portion Z is generally a polypeptide comprising about 2 up to about 15amino acid residues in length. In some embodiments, portion Z is up toabout 5, up to about 10, up to about 15, up to about 20, up to about 25,up to about 30, up to about 35, up to about 40, up to about 45, or up toabout 50 amino acids in length. In some embodiments, portion Z rangesfrom about 2 to about 5, about 5 to about 10, about 10 to about 15,about 15 to about 20, about 20 to about 25, about 25 to about 30, about30 to about 35, about 35 to about 40, about 40 to about 45, or about 45to about 50 amino acids in length. Portion Z is no more than about 55amino acids in length.

Portion Z may comprise any type of amino acid residue. In someembodiments, portion Z may optionally comprise a detectable moiety tofacilitate detection of portion Z following cleavage of X².

Recognition Domains

Portion Z may be designed to aid in modulating the cleavage of X² andsubsequent activation of portion A. In some embodiments, portion Zcomprises an amino acid sequence that can be bound by an enzyme thatcleaves X². In certain embodiments, recognition of a specific amino acidsequence in portion Z may be required before an enzyme is able to cleaveX² and activate the promolecule. Some of the amino acid residues inportion Z may be adjusted to modulate the activity of the promoleculewithout changing the specificity of interaction between portion Z andthe target enzyme. The crystal structure of the enzyme thrombin bound toportion Z of a promolecule is shown in FIG. 3. As shown in FIG. 4, thenumber of interactions between certain amino acid residues in portion Zand a target enzyme may be used to determine which amino acid residuescan altered without impacting binding of the enzyme to portion Z.

In some embodiments, portion Z comprises an amino acid sequence derivedfrom naturally occurring physiologic substrates of the enzymaticactivator. In some embodiments, portion Z comprises an amino acidsequence derived from the analysis of screening of combinatorial peptidelibraries that may or may not share similarity to physiologic substratesof the enzymatic activator.

In some embodiments, portion Z comprises the exosite recognitionsequence of protease-activated receptor-1 (PAR-1), which is specificallyrecognized by the enzyme thrombin. In some embodiments, portion Zcomprises the PAR-1 exosite recognition sequence SFLLRNPNDKYEPFW (SEQ IDNO: 18).

In some embodiments, portion Z comprises the exosite recognitionsequence of protease-activated receptor-2 (PAR-2), which is specificallyrecognized by the enzyme TMPRSS2. In some embodiments, portion Zcomprises the PAR-2 exosite recognition sequence KVDGTSHVTGDDD (SEQ IDNO: 20).

X²—Cleavable Linkers

In the promolecules of the present disclosure, portion A is linked toportion Z through cleavable linker X². In some embodiments, X² comprisesa linker that links portion A to portion Z with a single chemical bond.In other embodiments, X² comprises a chimeric linker that links portionA to portion Z through two or more different chemical bonds.

Cleavage of X² produces two cleavage products: a first cleavage productcontaining portion A and a second cleavage product containing portion Z.In general, X² is cleavable under a pre-selected physiologicalcondition. X² can be selected so that the promolecule is selectivelycleaved when exposed to an environment associated with a condition to bediagnosed or detected.

For example, X² may comprise a chemical bond that is susceptible tocleavage under conditions found in an extracellular environment, such asacidic conditions, which may be found near cancerous cells and tissues,or reducing environments, as may be found near hypoxic or ischemic cellsand tissues. X² may comprise a chemical bond that is subject to cleavageby proteases or other enzymes found on the surface of cells or releasednear cells having a condition to be diagnosed or detected, such asdiseased, apoptotic or necrotic cells and tissues, or by otherconditions or factors. An acid-labile linker may be, for example, acis-aconitic acid linker. Other examples of pH-sensitive linkagesinclude acetals, ketals, activated amides such as amides of2,3-dimethylmaleamic acid, vinyl ether, other activated ethers andesters such as enol or silyl ethers or esters, imines, iminiums,enamines, carbamates, hydrazones, and other linkages.

X² may comprise an amino acid or a peptide. When X² comprises a peptide,the peptide may be of any suitable length, such as, for example, about 2up to about 5, up to about 10, up to about 15, up to about 20, up toabout 25, or up to about 30 amino acid residues in length. In someembodiments, X² is about 2 to about 5, about 5 to about 10, about 10 toabout 15, about 15 to about 20, about 20 to about 25, or about 25 toabout 30 amino acids in length. X² is no longer than about 35 aminoacids in length. A cleavable peptide may include an amino acid sequencerecognized and cleaved by a protease, so that proteolytic action of theprotease cleaves X².

Enzymatically-Cleavable Linkers

The design of X² for cleavage by specific conditions, such as by aspecific enzyme, allows targeting of activation to a specific locationwhere such conditions are found. Thus, one way that compounds of thepresent disclosure provide specific targeting to desired cells, tissues,or regions is by the design of the linker portion X² to be cleaved byconditions near such targeted cells, tissues, or regions. After cleavageof X², cleavage products A and Z are formed, and portion A is free tointeract with phospholipid bilayers, such as cell membranes, in thevicinity of activation.

In some embodiments, X² is an enzymatically-cleavable peptide having theamino acid sequence PR. This amino acid sequence is specifically cleavedby the enzyme thrombin. In some embodiments, X² is anenzymatically-cleavable peptide having the amino acid sequence PLGLAG(SEQ ID NO: 24). This amino acid sequence is specifically cleaved by theenzyme matrix metalloproteinase-2 (MMP-2). In some embodiments, X² is anenzymatically-cleavable peptide having the amino acid sequence PLFAEP(SEQ ID NO: 25), which is cleaved by the enzyme calpain-1. In someembodiments, X² is an enzymatically-cleavable peptide having the aminoacid sequence GLGSEP (SEQ ID NO: 26), which is cleaved by the enzymecalpain-2. In some embodiments, X² is an enzymatically-cleavable peptidehaving the amino acid sequence KSRAEDE (SEQ ID NO: 28), which is cleavedby the enzyme matriptase. In some embodiments, X² is anenzymatically-cleavable peptide having the amino acid sequence LQRALE(SEQ ID NO: 29), which is cleaved by the enzyme MASP-2. In someembodiments, X² is an enzymatically-cleavable peptide having the aminoacid sequence LLRSLIG (SEQ ID NO: 30), which is cleaved by the enzymeTMPRSS2. In some embodiments, X² is an enzymatically-cleavable peptidehaving the amino acid sequence VELLYLV (SEQ ID NO: 31), which is cleavedby the secreted aspartyl proteases from a variety of species of Candidaand Aspergillus.

Linkers Cleavable by Thrombin or Other Clotting-Related Enzymes

In some embodiments, X² linkers are susceptible to cleavage by theenzyme thrombin, which is an enzyme involved with the process of bloodclot formation. Thrombin is present on the surface of cells in areas ofactive clotting, and may therefore be used to cleave X² linkers andtarget detection of active clotting using the promolecules of thepresent disclosure. Thrombin cleaves X² linkers that contain the aminoacid sequence PR, and also binds to the PAR-1 recognition sequence,which may be used to further control cleavage of X² linkers by thisenzyme. Promolecules of the present disclosure are cleaved by thrombinwhen an X² linker comprises the amino acid sequence PR, and when portionZ comprises the PAR-1 recognition sequence.

In some embodiments, X² linkers may be cleaved by other agentsassociated with the clotting process. For example, Factor Xa cleaveslinkers with the amino acid sequence LEGR (SEQ ID NO: 32). Factor IXacleaves linkers with the amino acid sequence LVVR (SEQ ID NO: 33).Activated protein C cleaves linkers with the amino acid sequence LVKR(SEQ ID NO: 36). Factor VIIa cleaves linkers with the amino acidsequence QLTR (SEQ ID NO: 37).

Matriptase-Cleavable Linkers

In some embodiments, X² linkers are susceptible to cleavage by theenzyme matriptase, which is an enzyme that may be over-expressed ininfiltrating breast carcinomas. Matriptase may therefore be used tocleave X² linkers and target detection of cancer, e.g. breast cancer,using the promolecules of the present disclosure. Matriptase cleaves X²linkers that contain the amino acid sequence KSR.

MMP-Cleavable Linkers

In some embodiments, X² linkers are susceptible to cleavage by matrixmetalloproteinases (MMPs). Thus, molecules having features of thepresent disclosure are able to direct membrane interaction of portion Awith specific cells, tissues, or regions having active MMPs in theextracellular environment.

For example, an X² linker that includes the amino acid sequence PLGLAG(SEQ ID NO: 24) may be cleaved by the metalloproteinase enzyme MMP-2 (amajor MMP in cancer and inflammation). Cleavage of such an X² linkeroccurs between the central G and L residues, resulting in separation ofportions A and Z, which in turn allows portion A to interact withphospholipid bilayers in the vicinity. Other examples of such X² linkersinclude the amino acid sequence AAA, which is cleavable by the enzymeMMP-11, the amino acid sequence GPSG (SEQ ID NO: 38), which is cleavableby the enzyme MMP-8, and the amino acid sequence GPAG (SEQ ID NO: 39),which is cleavable by the enzyme MMP-9.

X² linkers of the present disclosure may be designed to bepreferentially sensitive to particular subclasses of MMPs, or toindividual members of the large MMP family of proteinases. For example,in some embodiments, X² peptide sequences designed to be cleaved bymembrane-anchored MMPs are utilized because their activity remainslocalized to the outer surface of the expressing cell.

Calpain-Cleavable Linkers

In some embodiments, X² linkers are susceptible to cleavage by enzymesassociated with areas of necrosis. Such an X² linker includes one thatis susceptible to cleavage by calpains (e.g. calpain-1 or calpain-2) orother proteases that may be released from necrotic cells. Such cleavageof X² by calpains would release portion A from portion Z, allowingportion A to interact with the membranes of diseased cells andneighboring cells in the vicinity of necrotic cells or tissues.

For example, in some embodiments, X² is an enzymatically-cleavablepeptide having the amino acid sequence PLFAEP (SEQ ID NO: 25), which iscleaved by the enzyme calpain-1. In areas where calpain-1 is present(such as necrotic areas), X² is cleaved, releasing portion A fromportion Z and allowing portion A to interact with cell membranes in thevicinity.

In some embodiments, X² is an enzymatically-cleavable peptide having theamino acid sequence GLGSEP (SEQ ID NO: 26), which is cleaved by theenzyme calpain-2. In areas where calpain-2 is present (e.g., necroticareas), X² is cleaved, releasing portion A from portion Z and allowingportion A to interact with cell membranes in the vicinity.

In some embodiments, X² is an enzymatically-cleavable peptide having theamino acid sequence VGVF (SEQ ID NO: 27), which is cleaved by the enzymecalpain-3.

Other Enzymatically-Cleavable Linkers

In some embodiments, X² is an enzymatically-cleavable linker having theamino acid sequence SSLY (SEQ ID NO: 40), which is cleaved by ProstateSpecific Antigen (KLK3). In some embodiments, X² is anenzymatically-cleavable linker having the amino acid sequence ASN, whichis cleaved by the enzyme Legumain. In some embodiments, X² is anenzymatically-cleavable linker having the amino acid sequence RR, whichis cleaved by the enzyme Cathepsin B. In some embodiments, X² is anenzymatically-cleavable linker having the amino acid sequence SSR, whichis cleaved by Urokinase-type plasminogen activator. In some embodiments,X² is an enzymatically-cleavable linker having the amino acid sequenceLLRSLIG (SEQ ID NO: 30), which is cleaved by the enzyme TMPRSS2. In someembodiments, X² is an enzymatically-cleavable linker having the aminoacid sequence DDDDK (SEQ ID NO: 41), which is cleaved by the enzymeenteropeptidase. In some embodiments, X² is an enzymatically-cleavablelinker having the amino acid sequence KKLK (SEQ ID NO: 42), which iscleaved by the enzyme Cruzain. In some embodiments, X² is anenzymatically-cleavable linker having the amino acid sequence QRQR (SEQID NO: 43), which is cleaved by the enzyme Complement Clr. In someembodiments, X² is an enzymatically-cleavable linker having the aminoacid sequence NISH (SEQ ID NO: 44), which is cleaved by the enzyme C5apeptidase. In some embodiments, X² is an enzymatically-cleavable linkerhaving the amino acid sequence VELLYLV (SEQ ID NO: 31), which is cleavedby the enzyme Aspartyl peptidase. In some embodiments, X² is anenzymatically-cleavable linker having the amino acid sequence PLG, whichis cleaved by the enzyme Cathepsin L. In some embodiments, X² is anenzymatically-cleavable linker having the amino acid sequence RSKR (SEQID NO: 45), which is cleaved by the enzyme Protein Convertase 5. Asummary of the above-listed enzymes, their cleavage sequences, and theirassociated conditions is provided in Table 3.

Linkers Susceptible to Cleavage Under Reducing Conditions

X² linkers that are susceptible to cleavage under reducing conditionsprovide for cleavage of promolecules of the present disclosure inregions having reduced oxygen concentration, such as regions surroundingcancer cells and cancerous tissues, infarct regions, and other hypoxicregions. Examples of cleavable linkers susceptible to cleavage underhypoxic conditions include those containing a disulfide bond. In ahypoxic environment, free thiols and other reducing agents becomeavailable extracellularly, while the oxygen that normally maintains theextracellular environment in an oxidizing state is depleted. This shiftin the redox balance promotes reduction and cleavage of a disulfide bondwithin an X² linker. In addition to disulfide linkages that takeadvantage of thiol-disulfide equilibria, linkages including quinonesthat are cleaved when reduced to hydroquinones may be used in an X²linker designed for cleavage in reducing environments.

pH-Sensitive Linkers

In some embodiments, X² linkers are designed to be cleaved in acidicenvironments, such as sites near damaged or hypoxic tissue. X² linkersthat are cleaved in acidic environments can be utilized to targetactivation of the promolecules of the present disclosure to acidicregions. Such targeting could be achieved with an acid-labile X² linker(e.g., by including in X² an acetal or vinyl ether linkage, or anotherlinkage that is cleaved under acidic conditions).

Combinations of Multiple Linkers

In some embodiments, X² comprises an amino acid sequence that providestwo or more sites susceptible to cleavage (e.g., by an enzyme). Where amolecule having features of the present disclosure includes an X² linkercomprising multiple cleavage sites, separation of portion A from portionZ may require cleavage of multiple bonds within the X² linker, which maytake place either simultaneously or sequentially. Such X² linkers mayinclude bonds having different chemical properties or cleavagespecificities, so that separation of portion A from portion Z requiresthat more than one condition or environment (“extracellular signals”) beencountered by the molecule before activation takes place. The cleavagesites may be the same or different, and where different may be referredto herein as a “chimeric” linker. Cleavage of chimeric X² linkers thusserves as a detector of combinations of such extracellular signals.

Chimeric X² linkers may be used to further modulate the targeting ofportion A to desired cells, tissue or regions. Boolean combinations ofextracellular signals can be used to broaden or narrow the conditionsunder which cleavage of X² occurs. Where chimeric X² linkers are used tolink portion A to portion Z, the different chemical bonds within thechimeric linker can be arranged in parallel or in series. When arrangedin parallel, the cleavage conditions are narrowed, since each bond mustbe cleaved before portion A may separate from portion Z. When thechemical bonds within the chimeric linker are arranged in series, thecleavage conditions are broadened, since cleavage of any one of thechemical bonds will result in separation of portion A from portion Z.

For example, in order to detect either a protease or hypoxia (i.e., tocleave X² in the presence of either a protease or hypoxia), a chimericX² linker is designed with the protease-sensitive andreduction-sensitive chemical bonds in series, so that cleavage of eitherbond would suffice to allow separation of portion A from portion Z.

Alternatively, in order to detect the presence of both a protease andhypoxia (i.e., to cleave X² in the presence of both a protease andhypoxia but not in the presence of only one of these conditions alone),a dual X² linker could be designed, e.g., a chimeric linker, to placethe protease sensitive bond between at least one pair of cysteines thatare disulfide-bonded to each other (i.e., the chemical bonds in thechimeric X² linker are arranged in parallel). In this case, bothprotease cleavage and disulfide reduction are required in order to allowseparation of portions A and Z.

In certain embodiments, promolecules of the present disclosure may havethe following formula (I), wherein portion X² comprises a dual linker,which may be a chimeric linker, and has a cyclic structure.

wherein X^(1a), A, Z, and X^(1b) are as described herein. In formula (I)above, X^(2a) and X^(2b) are amino acids of X², wherein X^(2a) andX^(2b) may be independently selected from any amino acid; X^(2c) andX^(2d) comprise amino acids which provide a cleavable linker (e.g., anenzymatically cleavable linker); n is one or two; m and o are at leastone, and may be independently selected from an integer ranging from 1 to30; p and q are each at least two, and may be independently selectedfrom an integer ranging from two to thirty, wherein the cleavage sitesprovided by X^(2c) and X^(2d) may the same or different, may besusceptible to cleavage by the same or different conditions (e.g., thesame or different enzymes), and may be, for example, independentlyselected from any of the cleavable linkers described herein. WhereX^(2c) and X^(2d) define cleavable linkers that are each susceptible tocleavage under different conditions (e.g., different enzymes), themolecule can be described as comprising a chimeric linker. Suchcombinations may include enzymes of the same or different class. Forexample, pairing cleavage sequences of thrombin with that of activatedprotein C may find utility in discriminating the driving forces betweenclot formation and its dissolution. For example, pairing the cleavagesequence of MMP-1 with an acid cleavable linker may find utility intargeting the probe to the hypoxic core of cancerous tumors.

Synthesis of a molecule of formula (I) can be performed with standardpeptide coupling chemistry. For example, as a precursor to compounds offormula (I), standard peptide coupling chemistry can be used to make acompound of the formula (II) below, wherein X² comprises an asparticacid or glutamic acid and lysine residues.

A peptide coupling reaction typically employs a conventional peptidecoupling reagent and is conducted under conventional coupling reactionconditions, typically in the presence of a trialkylamine, such asethyldiisopropylamine or diisopropylethylamine (DIEA). Suitable couplingreagents for use include, by way of example, carbodiimides, such asethyl-3-(3-dimethylamino)propylcarbodiimide (EDC),dicyclohexylcarbodiimide (DCC), diisopropylcarbodiimide (DIC) and thelike, and other well-known coupling reagents, such asN,N′-carbonyldiimidazole, 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline(EEDQ), benzotriazol-1-yloxy-tris(dimethylamino)phosphoniumhexafluorophosphate (BOP),O-(7-azabenzotriazol-1-yl)-N,N,N,N′,N′-tetramethyluroniumhexafluorophosphate (HATU) and the like. Optionally, well-known couplingpromoters, such N-hydroxysuccinimide, 1-hydroxybenzotriazole (HOBT),1-hydroxy-7-azabenzotriazole (HOAT), N,N-dimethylaminopyridine (DMAP)and the like, can be employed in this reaction. Typically, this couplingreaction is conducted at a temperature ranging from about 0° C. to about60° C. for about 1 to about 72 hours in an inert diluent, such as THF orDMF.

During any of the processes for preparation of the compounds, it may benecessary and/or desirable to protect sensitive or reactive groups onany of the molecules concerned. This can be achieved by means ofconventional protecting groups as described in standard works, such asT. W. Greene and P. G. M. Wuts, “Protective Groups in OrganicSynthesis”, Fourth edition, Wiley, N.Y. 2006. The protecting groups canbe removed at a convenient subsequent stage using methods known in theart. For example, the aspartic acid, glutamic acid, and lysine residuescan be protected with various protecting groups during the syntheticprocess. Depending on the type of protecting group used, selectivity indeprotection can be used advantageously during the synthetic process.One of ordinary skill in the art would be able to select the type ofprotecting group that is appropriate for the synthetic scheme.

The dual linker described above having a cyclic structure can besynthesized by using the carboxyl side chain of aspartic acid orglutamic acid as the carboxyl handle of a peptide backbone and aminoside chain of lysine as the amino handle of a peptide backbone.Synthesis of formula (I) can be performed using standard peptidecoupling chemistry. Peptide coupling reactions typically employ aconventional peptide coupling reagent and are conducted underconventional coupling reaction conditions as discussed above.

Portions X^(1A) and X^(1B)

Promolecules of the present disclosure may include optional portionsX^(1a) and X^(1b) that, when present, comprise a nucleophilic moiety andfacilitate the attachment of one or more cargo moieties to thepromolecule. The nucleophilic moiety of portions X^(1a) and X^(1b)generally comprises a nucleophilic reactive group comprising at leastone pair of free electrons that is capable of reacting with anelectrophile. Examples of nucleophilic moieties include sulfurnucleophiles, such as thiols, thiolate anions, anions ofthiolcarboxylate, anions of dithiocarbonates, and anions ofdithiocarbamates; oxygen nucleophiles, such as hydroxide anion,alcohols, alkoxide anions, and carboxylate anions; nitrogennucleophiles, such as amines, azides, and nitrates; and carbonnucleophiles, such as alkyl metal halides and enols.

Cargo moieties may be, e.g., detectable moieties that can facilitatedetection of a promolecule through various imaging modalities, or maybe, e.g., therapeutic agents that can facilitate treatment of a diseaseor condition.

Non-limiting examples of detectable moieties include fluorescent dyesand radioisotopes. In some embodiments, two or more cargo moieties maybe attached to the same promolecule (e.g., a fluorescent dye and aradioisotope attached to the same promolecule). Differing cargo moietiesmay be paired for simultaneous detection using multiple modalities. Forexample, non-invasive detection using nuclear imaging agents could becoupled with fluorescence to enable follow on studies for enhanced yetinvasive (e.g., surgical) detection.

In some embodiments, a detectable moiety may comprise a fluorescent dye.Non-limiting examples of fluorescent dyes that may be conjugated topromolecules of the present disclosure include cyanine dyes, such asfluorescein, tetramethoxyrhodamine, Cy2, Cy3, Cy3B, Cy3.5, Cy5, Cy5.5,or Cy7, IRdye 800 cw, or ATTO-TEC™ dyes, such as ATTO 680. Suitablecargo moieties also include fluorescent dyes having longer wavelengthsin the near-infrared region. Such dyes are known in the art and can bereadily incorporated into the compounds of the present disclosure.

In some embodiments, a detectable moiety may comprise a radioisotope,e.g., a radioisotope chelated through a metal binding moiety.Non-limiting examples of radioisotopes include Calcium-47, Carbon-11,Carbon-14, Chromium-51, Cobalt-57, Cobalt-58, Erbium-169, Fluorine-18,Gallium-67, Gallium-68, Hydrogen-3, Indium-111, Iodine-123, Iodine-125,Iodine-131, Iron-59, Krypton-81m, Nitrogen-13, Oxygen-15, Phosphorus-32,Samarium-153, Selenium-75, Sodium-22, Sodium-24, Strontium-89,Technetium-99m, Thallium-201, Xenon-133, or Yttrium-90.

In some embodiments, a single promolecule having features of the presentdisclosure may include more than one cargo moiety so that portion A maybe linked to multiple detectable moieties, or to both a detectablemoiety and a therapeutic agent, or to multiple therapeutic agents. Suchmultiple detectable moieties may include different types of markers, andmay allow, for example, attachment of both a radioisotope and a contrastagent or fluorescent dye, allowing imaging by different modalities.

Promolecules comprising a detectable moiety conjugated through portionX^(1a) or X^(1b) may have use in visualization or identification ofcells having a certain condition or cells in a region exhibiting aparticular condition. For example, thrombosis (clot formation) may bevisualized by designing an X² linker to be cleaved by any of the manyproteases in the blood clot formation cascade, such as thrombin, so thata cleavage product comprising portion A interacts with cell membranes inthe vicinity of the clotting activity. Similarly, complement activationmay be visualized by designing an X² linker to be cleaved by any one ormore of the proteases in the complement activation cascades for deliveryof a fluorescent dye or other marker to the region. Thus, fluorescentmoieties are one example of a cargo moiety that may be delivered totarget cell membranes or phospholipid bilayer structures in specificregions upon cleavage of X².

Non-limiting examples of therapeutic agents that can be conjugated tothe promolecules of the present disclosure include radioisotopes such asCalcium-47, Carbon-11, Carbon-14, Chromium-51, Cobalt-57, Cobalt-58,Erbium-169, Fluorine-18, Gallium-67, Gallium-68, Hydrogen-3, Indium-111,Iodine-123, Iodine-125, Iodine-131, Iron-59, Krypton-81m, Nitrogen-13,Oxygen-15, Phosphorus-32, Samarium-153, Selenium-75, Sodium-22,Sodium-24, Strontium-89, Technetium-99m, Thallium-201, Xenon-133, orYttrium-90.

In some embodiments, a particular moiety may function as both adetectable moiety and as a therapeutic agent.

Methods of Making

Promolecules of the present disclosure can be made by any suitablemethod, including but not limited to recombinant and non-recombinant(e.g., chemical synthesis) methods. Cargo moieties may be conjugated topromolecules by any suitable method, including but not limited tonucleophilic addition reactions.

Production of Promolecules

The promolecules of the present disclosure can be produced by anysuitable method, including recombinant and non-recombinant methods(e.g., chemical synthesis).

Where a polypeptide is chemically synthesized, the synthesis may proceedvia liquid-phase or solid-phase. Solid-phase synthesis (SPPS) allows theincorporation of unnatural amino acids, peptide/protein backbonemodification. Various forms of SPPS, such as Fmoc and Boc, are availablefor synthesizing peptides of the present disclosure. Details of thechemical synthesis are known in the art (e.g., Ganesan A. 2006 Mini Rev.Med Chem. 6:3-10 and Camarero J A et al. 2005 Protein Pept Lett.12:723-8). Briefly, small insoluble, porous beads are treated withfunctional units on which peptide chains are built. After repeatedcycling of coupling/deprotection, the free N-terminal amine of asolid-phase attached peptide or amino acid is coupled to a singleN-protected amino acid unit. This unit is then deprotected, revealing anew N-terminal amine to which a further amino acid may be attached. Thepeptide remains immobilized on the solid-phase and undergoes afiltration process before being cleaved off.

Where the polypeptide is produced using recombinant techniques, theproteins may be produced as an intracellular protein or as a secretedprotein, using any suitable construct and any suitable host cell, whichcan be a prokaryotic or eukaryotic cell, such as a bacterial (e.g. E.coli) or a yeast host cell, respectively.

Other examples of eukaryotic cells that may be used as host cellsinclude insect cells, mammalian cells, and/or plant cells. Wheremammalian host cells are used, the cells may include one or more of thefollowing: human cells (e.g. HeLa, 293, H9 and Jurkat cells); mousecells (e.g., X3, NIH3T3, pancreatic ductal adenocarcinoma 2.1, L cells,and C127 cells); primate cells (e.g. Cos 1, Cos 7 and CV1) and hamstercells (e.g., Chinese hamster ovary (CHO) cells).

A wide range of host-vector systems suitable for the expression of thesubject polypeptide may be employed according to standard proceduresknown in the art. See, e.g., Sambrook et al. 1989 Current Protocols inMolecular Biology Cold Spring Harbor Press, New York and Ausubel et al.1995 Current Protocols in Molecular Biology, Eds. Wiley and Sons.Methods for introduction of genetic material into host cells include,for example, transformation, electroporation, conjugation, calciumphosphate methods and the like. The method for transfer can be selectedso as to provide for stable expression of the introducedpolypeptide-encoding nucleic acid. The polypeptide-encoding nucleic acidcan be provided as an inheritable episomal element (e.g., a plasmid) orcan be genomically integrated. A variety of appropriate vectors for usein production of a polypeptide of interest are available commercially.

Vectors can provide for extrachromosomal maintenance in a host cell orcan provide for integration into the host cell genome. The expressionvector provides transcriptional and translational regulatory sequences,and may provide for inducible or constitutive expression, where thecoding region is operably linked under the transcriptional control ofthe transcriptional initiation region, and a transcriptional andtranslational termination region. In general, the transcriptional andtranslational regulatory sequences may include, but are not limited to,promoter sequences, ribosomal binding sites, transcriptional start andstop sequences, translational start and stop sequences, and enhancer oractivator sequences. Promoters can be either constitutive or inducible,and can be a strong constitutive promoter (e.g., T7, and the like).Expression constructs generally have convenient restriction siteslocated near the promoter sequence to provide for the insertion ofnucleic acid sequences encoding proteins of interest. A selectablemarker operative in the expression host may be present to facilitateselection of cells containing the vector. In addition, the expressionconstruct may include additional elements. For example, the expressionvector may have one or two replication systems, thus allowing it to bemaintained in organisms, for example in mammalian or insect cells forexpression and in a prokaryotic host for cloning and amplification. Inaddition the expression construct may contain a selectable marker geneto allow the selection of transformed host cells. Selectable genes arewell known in the art and will vary with the host cell used.

Isolation and purification of a protein and/or antibody can beaccomplished according to methods known in the art. For example, aprotein can be isolated from a lysate of cells genetically modified toexpress the protein constitutively and/or upon induction, or from asynthetic reaction mixture, by immunoaffinity purification, whichgenerally involves contacting the sample with an anti-protein antibody,washing to remove non-specifically bound material, and eluting thespecifically bound protein. The isolated protein can be further purifiedby dialysis and other methods normally employed in protein purificationmethods. In one embodiment, the protein may be isolated using metalchelate chromatography methods. Protein of the present disclosure maycontain modifications to facilitate isolation.

The subject polypeptides may be prepared in substantially pure orisolated form (e.g., free from other polypeptides). The protein can bepresent in a composition that is enriched for the polypeptide relativeto other components that may be present (e.g., other polypeptides orother host cell components). Purified protein may be provided such thatthe protein is present in a composition that is substantially free ofother expressed proteins, e.g., less than 98%, less than 95%, less than90%, less than 80%, less than 60%, or less than 50%, of the compositionis made up of other expressed proteins.

Conjugation of Cargo Moieties to Polypeptides

Cargo moieties may be conjugated to promolecules of the presentdisclosure using any suitable technique, including but not limited tonucleophilic addition reactions that utilize nucleophilic moieties.Non-limiting examples of such reactions include reactions of sulfurnucleophiles, oxygen nucleophiles, carbon nucleophiles, or nitrogennucleophiles with a suitable electrophile to form a covalent bond.

Optional Modifications

Promolecules of the present disclosure may be further modified togenerally provide, e.g., longer circulating half-life, restriction ofthe promolecules to certain anatomical compartments (e.g., restrictionto the cardiovascular system), protection against non-specificdegradation, and/or enhanced sensitivity to certain imaging modalities.

In some embodiments, two or more promolecules may be linked to a centralmolecule, e.g., a polyethylene glycol (PEG) molecule, to form adendrimer using techniques that are known in the art. Suitable PEGmolecules may have a molecular weight of up to about 1,000, up to about5,000, up to about 10,000, up to about 20,000, up to about 30,000, or upto about 40,000 Daltons. Conjugation of two or more promolecules to acentral PEG molecule can be accomplished by, e.g., activating a PEGmolecule with a functional group at one or more termini and thenreacting the activated PEG molecule with one or more promolecules of thepresent disclosure. The choice of functional groups depends on theavailable reactive groups on the promolecule, such as the N-terminalamine, the C-terminal carboxylic acid, or residues such as lysine,aspartic acid, cysteine, glutamic acid, serine, threonine, or otherspecific reactive sites. Linear, single-arm PEG structures, as well asbranched PEG structures may be created using such techniques.

In some embodiments, a linear, single-arm PEG structure is formed havingthe general formula: X^(1a)-A-X²-Z-X³, where X³ is a PEG molecule andX^(1a), A, X², and Z are as described above. In other embodiments, abranched PEG dendrimer is formed using techniques known in the art,wherein two or more promolecules of the present disclosure areconjugated to the branched PEG dendrimer to form a polyvalent PEGstructure.

Formulations

Promolecules of the present disclosure can be formulated in a variety ofpharmaceutical compositions suitable for administration to a subject(e.g., by a desired route). A composition comprising a promolecule ofthe present disclosure may comprise a pharmaceutically acceptableexcipient, a variety of which are known in the art and need not bediscussed in detail herein.

In some embodiments, promolecules of the present disclosure areformulated for parenteral administration to a subject, e.g., intravenousadministration. Pharmaceutically acceptable excipients have been amplydescribed in a variety of publications, including, for example,“Remington: The Science and Practice of Pharmacy”, 19th Ed. (1995), orlatest edition, Mack Publishing Co; A. Gennaro (2000) “Remington: TheScience and Practice of Pharmacy”, 20th edition, Lippincott, Williams, &Wilkins; Pharmaceutical Dosage Forms and Drug Delivery Systems (1999) H.C. Ansel et al., eds 7th ed., Lippincott, Williams, & Wilkins; andHandbook of Pharmaceutical Excipients (2000) A. H. Kibbe et al., eds.,3rd ed. Amer. Pharmaceutical Assoc.

In some cases, a subject pharmaceutical composition will be suitable forinjection into a subject, e.g., will be sterile. For example, in someembodiments, a subject pharmaceutical composition will be suitable forinjection into a human subject, e.g., where the composition is sterileand is free of detectable pyrogens and/or other toxins.

A subject pharmaceutical composition may comprise other components, suchas pharmaceutical grades of mannitol, lactose, starch, magnesiumstearate, sodium saccharin, talcum, cellulose, glucose, sucrose,magnesium, carbonate, and the like. The compositions may containpharmaceutically acceptable auxiliary substances as required toapproximate physiological conditions such as pH-adjusting and bufferingagents, tonicity-adjusting agents and the like, for example, sodiumacetate, sodium chloride, potassium chloride, calcium chloride, sodiumlactate, hydrochloride, sulfate salts, solvates (e.g., mixed ionicsalts, water, organics), hydrates (e.g., water), and the like.

Promolecules of the present disclosure may be formulated into unitdosage forms that contain a predetermined amount of the promoleculesdisclosed herein. Unit dosage forms suitable for injection orintravenous administration may comprise promolecules of the presentdisclosure in a composition as a solution in sterile water, normalsaline, or another pharmaceutically acceptable carrier.

The term “unit dosage form,” as used herein, refers to physicallydiscrete units suitable as unitary dosages for human and animalsubjects, each unit containing a predetermined quantity of promoleculesof the present disclosure calculated in an amount sufficient to producethe desired effect in association with a pharmaceutically acceptablediluent, carrier or vehicle. The specifications for the unit dosageforms of the present disclosure depend on the particular promoleculeemployed and the effect to be achieved, and the pharmacodynamicsassociated with each promolecule in the subject.

Pharmaceutically acceptable excipients, such as vehicles, adjuvants,carriers or diluents, are readily available to the public. Moreover,pharmaceutically acceptable auxiliary substances, such as pH adjustingand buffering agents, tonicity adjusting agents, stabilizers, wettingagents and the like, are readily available to the public.

Promolecules of the present disclosure may also be formulated for oraladministration to a patient. For oral preparations, promolecules can beused alone or in combination with appropriate additives to make tablets,powders, granules or capsules, for example, with conventional additives,such as lactose, mannitol, corn starch or potato starch; with binders,such as crystalline cellulose, cellulose derivatives, acacia, cornstarch or gelatins; with disintegrators, such as corn starch, potatostarch or sodium carboxymethylcellulose; with lubricants, such as talcor magnesium stearate; and if desired, with diluents, buffering agents,moistening agents, preservatives and flavoring agents.

The promolecules of the present disclosure may be utilized in aerosolformulations to be administered via inhalation, or may be formulatedinto acceptable pressurized propellants, such asdichlorodifluoromethane, propane, nitrogen, and the like.

Furthermore, promolecules of the present disclosure can be made intosuppositories by mixing with a variety of bases such as emulsifyingbases or water-soluble bases. The promolecules of the present inventioncan be administered rectally via a suppository. The suppository caninclude vehicles such as cocoa butter, carbowaxes, and polyethyleneglycols, which melt at body temperature, yet are solidified at roomtemperature.

Unit dosage forms for oral or rectal administration such as syrups,elixirs, and suspensions may be provided wherein each dosage unit, forexample, teaspoonful, tablespoonful, tablet or suppository, contains apredetermined amount of the promolecules of the present disclosure.Similarly, unit dosage forms for injection or intravenous administrationmay comprise one or more promolecules in a composition as a solution insterile water, normal saline or another pharmaceutically acceptablecarrier.

In some embodiments, promolecules of the present disclosure areformulated for local administration to a subject, e.g., at or near asite of desired action. In some embodiments, promolecules of the presentdisclosure are formulated in a sustained release dosage form that isdesigned to release promolecules at a predetermined rate for a specificperiod of time.

Promolecules of the present disclosure may also be formulated withagents that influence the pharmacokinetic profile of the promoleculewhen administered to a subject. Such agents include verapamil or otherequivalents.

Routes of Administration

In practicing the methods of the present disclosure, routes ofadministration may be selected according to any of a variety of factors,such as properties of the promolecule to be delivered, the type ofcondition being diagnosed, detected, or treated (e.g., detection ofclotting), and the like. Promolecules of the present disclosure may bedelivered by a route of administration that provides delivery of thepromolecule to the bloodstream (e.g., by parenteral administration, suchas intravenous administration, intramuscular administration, and/orsubcutaneous administration) or to a specific tissue or organ (e.g.,muscle tissue, cardiac tissue, vascular tissue, and the like). Injectioncan be used to accomplish parenteral administration. In someembodiments, promolecules are delivered by a route of administrationthat provides for delivery of the promolecule directly into affectedtissue, e.g., by direct injection into the target tissue or organ.

Promolecules of the present disclosure may be administered through therespiratory tract. Such dosage forms may be smoking devices, dry powderinhalers, pressurized metered dose inhalers, nebulizers, vaporizers, orthe like.

Promolecules of the present disclosure may be administered orally byhaving the subject swallow a suitable dosage form, such as tablets,powders, granules, capsules, elixirs, syrups, or the like. Promoleculesof the present disclosure may also be administered rectally in the formof suppositories.

Promolecules of the present disclosure may be administered by directinjection into a target tissue or into the blood stream, includingintradermal, subcutaneous, intravenous, intracardiac, intramuscular,intraosseous, or intraperitoneal injection. Promolecules of the presentdisclosure can be administered by intracavernous or intravitrealdelivery to organs or tissues, or administered by intracerebral,intrathecal, or epidural delivery to tissues of the central nervoussystem.

Promolecules of the present disclosure may be administered locally ortopically. Such administration may be accomplished by topically applyinga suitable formulation directly to a target tissue. Thepreviously-described routes of administration, formulations and dosageforms are merely exemplary and are in no way limiting.

Dosages

In the methods of the present disclosure, an amount of a promoleculethat is effective to achieve the desired diagnosis, detection, ortreatment is administered to a subject.

The amount administered varies depending upon the goal of theadministration, the health and physical condition of the individual tobe treated, age, the degree of resolution desired, the formulation of asubject composition, the activity of the subject composition employed,the treating clinician's assessment of the medical situation, thecondition of the subject, the body weight of the subject, as well as theseverity of the disease, disorder, or condition being diagnosed,detected, and/or treated, and other relevant factors. The size of thedose will also be determined by the existence, nature, and extent of anyadverse side-effects that might accompany the administration of aparticular composition.

It is expected that the amount will fall in a relatively broad rangethat can be determined through routine trials. For example, the amountof a promolecule of the present disclosure employed to detect activeclotting in a subject is not more than about the amount that couldotherwise be irreversibly toxic to the subject (i.e., maximum tolerateddose). In other cases, the amount is around or even well below the toxicthreshold, but still in an effective concentration range, or even as lowas a threshold dose. In some embodiments, a dose of 100 micrograms isadministered to a subject to detect blood clots in deep vein thrombosis.

Methods of Use

The present disclosure provides methods of using activatable anddetectable membrane-interacting peptides for the diagnosis and/ortreatment of diseases or conditions generally involving localizedbiological processes, such as proteolysis. For example, in certaincases, the promolecules of the present disclosure find use as adiagnostic tool in guiding and/or monitoring therapy. Such methodsgenerally involve detection of biological processes, such asproteolysis, which may be associated with a particular disease orcondition. The promolecules of the present disclosure also find use intreating particular diseases or conditions, and in methods that involvedelivery of therapeutic agents to a particular site or location within apatient.

In general, the methods of the present disclosure involve selecting apromolecule that contains a cleavable linker X² that is cleaved underconditions associated with a disease or condition to be diagnosed,detected, or treated, and administering the promolecule to a subject inan amount that is sufficient to facilitate detection of thecleavage-promoting condition(s) or to facilitate treatment of adisease/condition associated with the cleavage-promoting condition(s).Administering can be by any suitable route, and the promolecule can beselected according to the disease or condition to be diagnosed ortreated. For example, in the case of detection of active clotting withina subject, administration can be intravenous.

When the promolecule encounters cleavage-promoting conditions within thesubject, the promolecule is cleaved and the cleavage product containingthe membrane-interacting peptide inserts into membranes in the vicinityof the cleavage-promoting environment. In diagnostic uses and methods,identification of the region within the subject where cleavage promotingconditions exist facilitates diagnosis of a disease or condition, andmay then provide guidance for administering and/or monitoring anappropriate therapy. In therapeutic uses and methods, delivery of atherapeutic agent to a targeted site within the patient facilitatestreatment of a disease or condition.

Diagnostic Uses and Methods

The methods of the present disclosure generally relate to diagnosis anddetection of diseases or conditions that involve localized biologicalprocesses, such as proteolysis. In some embodiments, the methods of thepresent disclosure relate to detecting proteolysis resulting from theactivity of one or more enzymes that are associated with a particularcondition. Such enzymes may be, e.g., bound to or associated with cellslocated in particular tissues or organs, and the identification of suchcells may be useful in guiding and/or monitoring therapy. For example,the enzyme thrombin is associated with sites of active clotting, and theidentification of such sites may be useful in diagnosing conditions suchas a ruptured arterial plaque. Other enzymes, such as, e.g., matrixmetalloproteinase-2 (MMP-2) are known to be associated withmalignancies, and their identification may be useful in diagnosingcancer and monitoring tumor growth. In other embodiments, the methods ofthe present disclosure involve diagnosing infection by detectingproteolysis caused by enzymes associated with infectious organisms,e.g., bacteria or fungi. In such embodiments, the methods of the presentdisclosure involve detection of proteolysis caused by proteases secretedby an infectious organism, which facilitates diagnosis of the infection.

In other embodiments, the methods of the present disclosure relate todiagnosing conditions that give rise to particular extracellularenvironments, such as, e.g., inflammation. For example, conditions thatgive rise to acidic extracellular environments may be detected usingpromolecules of the present disclosure that contain acid-labile linkers.Similarly, conditions that give rise to reducing extracellularenvironments may be detected using promolecules of the presentdisclosure containing linkers that are cleavable under reducingconditions, e.g., disulfide linkers, which may facilitate guidance andmonitoring of therapy for such conditions. Non-limiting example methodsof the present disclosure are provided below.

The methods of the present disclosure can be adapted to provide formethods of monitoring therapy. For example, a promolecule of the presentdisclosure can be administered to a subject prior to, during (e.g.,between doses), and/or after therapy, and the signal associated with thepromolecule can be detected to facilitate the effect of therapy upon thecondition being treated.

Detection of Active Clotting

In some embodiments, promolecules are administered to a subject todiagnose active clotting. For example, a promolecule having athrombin-cleavable X² linker and having a fluorescent cargo moietyconjugated thereto is administered intravenously to a subject suspectedof having a condition involving active clotting, e.g., internalbleeding, or a ruptured arterial plaque. When the promolecule comes intocontact with thrombin at a site of active clotting in the subject, X² iscleaved, forming cleavage products containing portions A and Z. Themembrane-interacting peptide of portion A then undergoes aconformational change to form an alpha-helical structure that insertsinto cell membranes in the area of the active clotting. The fluorescentcargo moiety attached to portion A can then be detected usingfluorescence microscopy to identify the tissues in the subject in whichactive clotting is taking place. Once these tissues have beenidentified, an appropriate therapy can be administered to the subject.

In some embodiments, the cargo moiety attached to the promolecule is aradioisotope. Once the cleavage product containing portion A hasinserted into the plasma membranes of cells in the area of activeclotting, as described above, the radioisotope is detected using anappropriate imaging modality, e.g., fluoroscopy, X-rays, single photonemission computed tomography (SPECT), magnetic resonance (MR) orpositron emission tomography (PET) to identify tissues in the subject inwhich active clotting is taking place.

In some embodiments, promolecules of the present disclosure areadministered to a subject during or after therapy in order to monitorthe progress of the therapy. For example, after angioplasty has beenperformed on a subject to treat a ruptured arterial plaque, promoleculesof the present disclosure are administered to the subject in order todetermine whether any active clotting is still taking place. Asdescribed above, a promolecule having a cleavable X² linker that iscleaved by thrombin and having a fluorescent cargo moiety or aradioisotope cargo moiety conjugated thereto is administeredintravenously to the subject. If there are still areas of activeclotting within the treated plaque, the promolecule will be cleaved bythrombin at the site, portion A will undergo a conformational change toform an alpha-helical structure and insert into cell membranes in thevicinity, and the active clotting activity can be detected byvisualizing the fluorescent moiety or radioisotope. If the clottingactivity has decreased, then a diminished signal will be detected at thesite of treatment. This information can then be used by the treatingphysician to monitor the progress of the therapeutic efforts.

Detection of Cancerous Tissues

In some embodiments, the promolecules of the present disclosure areadministered to a subject to identify cancerous tissues, e.g., tumors,so that appropriate therapeutic measures can be taken. For example, insome embodiments, a promolecule having a cleavable X² linker that iscleaved by an enzyme that is over-expressed in cancer cells (e.g.,matriptase) is administered to a subject. When the promolecule comesinto contact with cancerous cells that over-express a target enzyme, X²is cleaved, forming cleavage products containing portions A and Z. Themembrane-interacting peptide of portion A then undergoes aconformational change to form an alpha-helical structure that insertsinto cell membranes in the area of the cancerous cells. The fluorescentcargo moiety attached to portion A can then be detected usingfluorescence microscopy to identify the cancerous cells in the subject.Once the cancerous cells have been identified, an appropriate therapycan be administered to the subject, e.g., surgical removal of the tumor.

In some embodiments, a promolecule having a cleavable X² linker that iscleaved under reducing conditions (e.g. a thiol X² linker), as may beencountered in hypoxic environments surrounding cancerous tumors, isadministered intravenously to a subject. When the promolecule comes intocontact with reducing conditions, X² is cleaved, forming cleavageproducts containing portions A and Z. The membrane-interacting peptideof portion A then undergoes a conformational change to form analpha-helical structure that inserts into cell membranes in the area ofthe cancerous tumors. The fluorescent cargo moiety attached to portion Acan then be detected using fluorescence microscopy to identify thecancerous tissues in the subject. Once the cancerous tissues have beenidentified, an appropriate therapy can be administered to the subject,e.g., surgical removal of the tissues.

Conditions Amenable to Diagnosis

Any condition generally associated with a localized biological process(e.g., enhanced enzymatic activity) or environment (e.g., hypoxia) isamenable to diagnosis using the promolecules and methods disclosedherein. In general, a promolecule is selected to include an X² linkerportion having a structure that is cleavable under the conditionsassociated with the condition to be diagnosed. Non-limiting examples ofconditions amenable to diagnosis using the promolecules and methods ofthe present disclosure are provided below in Table 4.

TABLE 4 List of conditions amenable to diagnosis usingenzymatically-cleavable promolecules of the present disclosure. EnzymeEnzyme Associated Cleavage Condition with Condition Sequence ConditionsInvolving Clotting Heart attack (e.g. due to ruptured plaque); ThrombinPR Stroke; Thrombosis associated with surgical Factor IXa LVVRintervention; (SEQ ID NO: 33) Left atrial appendage thrombus; ActivatedProtein C LVKR Deep vein thrombosis; (SEQ ID NO: 36)Thrombosis-associated with cancer; Factor VIIa QLTR Trauma; (SEQ ID NO:37) Factor Xa LEGR (SEQ ID NO: 32) Cancer Cancer (all types) MMP-2PLGLAG (SEQ ID NO: 24) Breast cancer Prostate specific antigen SSLY(KLK3) (SEQ ID NO: 40) Legumain ASN Cathepsin B RR Stromelysin 3 AAA(MMP-11) Urokinase-type SSR plasminogen activator Matriptase KSRAEDE(SEQ ID NO: 28) Prostate cancer TMPRSS2 LLRSLIG (SEQ ID NO: 30) NecrosisCalpain-1 PLFAEP (SEQ ID NO: 25) Calpain-2 GLGSEP (SEQ ID NO: 26)Inflammation Arthritis MMP-8 GPSG (SEQ ID NO: 38) PancreatitisEnteropeptidase DDDDK (SEQ ID NO: 41) Infection Parasitic infectionCruzain KKLK (SEQ ID NO: 42) Bacterial infection Complement C1r QRQR(SEQ ID NO: 43) C5a peptidase NISH (SEQ ID NO: 44) Fungal infectionAspartyl protease VELLYLV (SEQ ID NO: 31) Neurodegenerative diseaseCathepsin L PLG Proprotein Convertase 5 RSKR (SEQ ID NO: 45) Nephropathy(e.g. diabetic) MMP-9 GPAG (SEQ ID NO: 39) Muscular degenerativediseases Calpain-3 VGVF (SEQ ID NO: 27)Methods of Detecting Cleavage Products A and Z in DiagnosticApplications

Cleavage products A and Z may be detected through a variety of imagingand detection modalities, including by not limited to fluorescencemicroscopy, X-rays, fluoroscopy, angiography, positron emissiontomography (PET), and the like. Detection can be accomplished bydirectly imaging the cells or tissues in which the cleavage product islocated, or by contacting the cells or tissues in which the cleavageproduct is located with a secondary molecule or reagent, such as anantibody, followed by imaging or detecting the secondary molecule orreagent.

In some embodiments, one or more of the cleavage products is conjugatedto a cargo moiety that facilitates detection. In some embodiments, thecargo moiety is a fluorescent dye. In such embodiments, the cargo moietymay be detected directly using fluorescence microscopy, wherein cells,tissues, or entire subjects are placed in the field of a fluorescencemicroscope and visualized directly.

In some embodiments, the cargo moiety is a radioisotope. In suchembodiments, the cargo moiety may be detected using X-rays, fluoroscopy,angiography, positron emission tomography (PET), or single positronemission computed tomography (SPECT) wherein cells, tissues, or entiresubjects are placed in the field of the imaging modality and visualized.

In some embodiments, cleavage products are detected using a secondarymolecule or reagent, e.g., an antibody, which specifically binds to orinteracts with the cleavage products, e.g., an antibody thatspecifically binds to amino acid sequences in the cleavage products. Insuch embodiments, cells or tissues containing a cleavage product arecontacted with a secondary molecule or reagent, followed by imaging ordetecting the secondary molecule or reagent.

Therapeutic Uses and Methods

The methods of the present disclosure generally relate to treatment ofdiseases or conditions that involve localized biological processes, suchas proteolysis. In general, treatment is accomplished by administering apromolecule having an attached therapeutic agent, e.g., a radioisotope,by a route that facilitates delivery of the therapeutic agent to targetcells and tissues within a patient. The following are provided asnon-limiting exemplary therapeutic uses and methods.

Treatment of Cancerous Tissues

In some embodiments, promolecules of the present disclosure find use inthe treatment of cancer by delivering therapeutic agents, e.g.,radioisotopes, to cancerous tissues within a patient. Selection of apromolecule for use in treatment is based on expression orover-expression of particular enzymes by cancer cells. For example, theenzyme MMP-2 (cleavage sequence PLGLAG (SEQ ID NO: 24)) is generallyexpressed by all cancer cells. Breast cancer cells are known to expressprostate specific antigen KLK3 (cleavage sequence SSLY (SEQ ID NO: 40)),legumain (cleavage sequence ASN), cathepsin B (cleavage sequence RR),stromelysin 3 (also known as MMP-11) (cleavage sequence AAA),urokinase-type plasminogen activator (cleavage sequence SSR), andmatriptase (cleavage sequence KSRAEDE (SEQ ID NO: 28)). Prostate cancercells are known to express TMPRSS2 (cleavage sequence LLRSLIG (SEQ IDNO: 30)). Alternatively or in addition, cancerous tissues may also beassociated with hypoxia or other generally localized conditions.

Cancers amenable to treatment are generally those surrounded by a tissueenvironment in which an enzyme is present at a localized concentrationrelative to normal, non-cancerous tissue and/or in which a localizedenvironment (e.g., a hypoxic environment) is present relative to normal,non-cancerous tissue. Such cancers can include solid and semi-solidtumors. Promolecules having a therapeutic agent attached as portionX^(1a) and a cleavable X² linker that is cleaved by one or more enzymesor conditions associated with a particular type of cancer may be used tofacilitate delivery of a therapeutic agent, e.g., a radioisotope, to thecancerous tissues within a patient. After administration of apromolecule to the patient, the cleavable X² linker is cleaved at ornear the site of desired action by an enzyme or condition associatedwith the cancerous tissue, and the cleavage product comprising portion Ainserts into cell membranes and delivers the therapeutic agent to thecancer cells. Delivery of the therapeutic agent to the cancer cellstreats the patient by, e.g., killing the cancerous cells, and/or slowingthe growth of the cancerous cells.

Screening Methods

The promolecules of the present disclosure generally find use inscreening methods, e.g., in vitro or in vivo screening of candidateagents for a desired activity. In some embodiments, the presentdisclosure relates to methods for screening cells in vitro, e.g., toassess activity of an enzyme expressed by the cells, or to screen forcandidate agents that modulate enzymatic activity in the cells. In otherembodiments, the present disclosure relates to in vivo screening methodsthat can be used, e.g., to screen candidate agents for a desiredactivity in transgenic animal models of disease.

In some embodiments, the screening methods of the present disclosureinvolve contacting cells in vitro with promolecules having X² linkersdesigned to be cleaved by an enzyme of interest. At the surface of cellsthat express the enzyme of interest, the promolecules are cleaved andthe cleavage product containing the membrane-interacting peptideundergoes a conformational change, typically forming an alpha-helicalstructure that interacts with the plasma membrane of the cell, therebylabeling the cells that express the enzyme of interest. A detectablemoiety conjugated to the cleavage product comprising themembrane-interacting peptide can then be detected, which facilitatesscreening for cells that express the enzyme of interest. The amount ofcleavage product that accumulates at a given location or position cantherefore be used to screen for a desired enzymatic activity.

In some embodiments, the present disclosure relates to methods forscreening cells in vitro that create a particular extracellularenvironment. In certain embodiments, promolecules are designed so thatX² is cleaved under certain extracellular conditions, e.g., acidicconditions and/or hypoxic conditions. Cultured cells are contacted withsuch promolecules, and the promolecules are activated by cells thatcreate acidic and/or hypoxic extracellular environments. After cleavageof X², the cleavage product containing the membrane-interacting peptideundergoes a conformational change to form an alpha-helical structurethat inserts into the plasma membrane of the cells, thereby labeling thecells that produce acidic and/or hypoxic extracellular conditions. Thedetectable moiety conjugated to the cleavage product comprising themembrane-interacting peptide can then be detected, which facilitatesscreening for cells that produce acidic and/or hypoxic conditions.

The methods of the present disclosure also relate to screening methodsthat can be used to identify candidate agents or test compounds having adesired activity, e.g., candidate agents that modulate enzymaticactivity. In some embodiments, a screening method involves culturingcells in vitro and contacting the cells with a candidate agent or testcompound. The cultured cells are then contacted with promolecules of thepresent disclosure comprising a cleavable linker that is cleaved by anenzyme of interest. Candidate agents or test compounds that elicit adesired activity in the cultured cells facilitate the production ofcleavage-promoting conditions that result in cleavage of X². Aftercleavage of X², the cleavage product comprising the membrane-interactingpeptide undergoes a conformational change to form an alpha-helicalstructure that inserts into the plasma membrane of the cells, therebylabeling the cells. An increase in the level of cellular labeling in thepresence of the candidate agent or test compound as compared to thelevel of cellular labeling in the absence of the candidate agent or testcompound indicates that the candidate agent or test compound has adesired activity.

The methods of the present disclosure also relate to methods ofscreening cells in vivo, e.g., to identify candidate agents or testcompounds having a desired activity, e.g., candidate agents thatmodulate enzymatic activity. For example, the screening methodsdiscussed above may be conducted in vivo in animal models, e.g.,transgenic animal models of disease, to identify cells or tissues ofinterest that express (or fail to express) a particular enzyme ofinterest, or to identify cells or tissues of interest that create aparticular extracellular environment, e.g., an acidic and/or hypoxicextracellular environment in response to the candidate agent or testcompound.

Kits

Also provided by the present disclosure are kits for using thepromolecules disclosed herein and for practicing the methods, asdescribed above. The kits may be provided for administration ofpromolecules to a subject in which a disease or condition are to bediagnosed. The kit can include one or more of the promolecules and/orcargo moieties as disclosed herein, which may be provided in a sterilecontainer, and can be provided in formulation with a suitablepharmaceutically acceptable excipient for administration to a subject.The promolecules can be provided in a formulation that is ready to beused as it is or can be reconstituted to have the desiredconcentrations. Where the promolecules are provided to be reconstitutedby a user, the kit may also provide buffers, pharmaceutically acceptableexcipients, and the like, packaged separately from the subjectpromolecules.

In addition to the above-mentioned components, the kits can furtherinclude instructions for using the components of the kit to practice themethods of the present disclosure. The instructions for practicing thesubject methods are generally recorded on a suitable recording medium.For example, the instructions may be printed on a substrate, such aspaper or plastic, etc. As such, the instructions may be present in thekits as a package insert, in the labeling of the container of the kit orcomponents thereof (i.e., associated with the packaging orsubpackaging), etc. In other embodiments, the instructions are presentas an electronic storage data file present on a suitable computerreadable storage medium, e.g. CD-ROM, diskette, etc. In yet otherembodiments, the actual instructions are not present in the kit, butmeans for obtaining the instructions from a remote source, e.g. via theinternet, are provided. An example of this embodiment is a kit thatincludes a web address where the instructions can be viewed and/or fromwhich the instructions can be downloaded. As with the instructions, thismeans for obtaining the instructions is recorded on a suitablesubstrate.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the present invention, and are not intended to limit thescope of what the inventors regard as their invention nor are theyintended to represent that the experiments below are all or the onlyexperiments performed. Efforts have been made to ensure accuracy withrespect to numbers used (e.g. amounts, temperature, etc.) but someexperimental errors and deviations should be accounted for. Unlessindicated otherwise, parts are parts by weight, molecular weight isweight average molecular weight, temperature is in degrees Celsius, andpressure is at or near atmospheric. Standard abbreviations may be used,e.g., bp, base pair(s); kb, kilobase(s); pl, picoliter(s); s or sec,second(s); min, minute(s); h or hr, hour(s); aa, amino acid(s); nt,nucleotide(s); i.m., intramuscular(ly); i.p., intraperitoneal(ly); s.c.,subcutaneous(ly); and the like.

Materials and Methods

The following materials and methods were used in the examples providedbelow.

Peptide Synthesis

Oligopeptides were created using the solid phase synthesis techniques ofMerrifield. In this approach, oligopeptides were built from repeatedcycles of coupling, washing, de-protection and then washing again. Ateach cycle, the free N-terminal amine of a solid-phase attached peptidewas coupled to a single N-protected amino acid unit whose subsequentde-protection revealed a new N-terminal amine to which a further aminoacid was attached. Peptide synthesis was used to create promolecules ofthe X^(1a)-A-X²-Z-X^(1b) structure or various portions thereof, e.g.,X^(1a)-A-X² . Conjugation at X^(1a) or X^(1b) to attach an imaging agentwas either (1) performed during the synthesis through the attachment ofa lysine unit whose amine was previously coupled to the detection agentor (2) attached to a cysteine on the N-terminus of the peptide usingthiol chemistry following synthesis and initial peptide purification. Inthe latter case, imaging agents such as the fluorescent dye Cy3, Cy5, orATTO 680 were added using their maleimide forms, which readily reactwith free thiols present on cysteine side chains to create a covalentlinkage. Peptides were synthesized by a commercial supplier and providedas lyophilized samples that were reconstituted in water with or withoutan organic solvent as necessary to solubilize the compound. In eachcase, the supplier provided reverse phase high-pressure liquidchromatograms and mass spectra to validate the material.

Analysis by Reverse Phase HPLC

Thrombin-mediated proteolysis of promolecules was performed byincubating the enzyme at concentrations ranging from 0.1 to 2 nM with a20 to 100 μM solution of the promolecule at 37° C. for up to three or afull twenty-four hours to ensure completeness. The extent of proteolysiswas assessed by reverse phase high-pressure liquid chromatography.Samples were run over a C₁₈ column using an acetonitrile gradient of 20%to 80% over a period of 30 minutes. Complete hydrolysis was evident bythe absence of the initial peak corresponding to the promolecule, andwas further validated using mass spectrometry.

Analysis by Intrinsic Fluorescence

Intrinsic fluorescence was used to characterize promolecules containingone or more tryptophan residues in portion A. As the spectral propertiesof tryptophan are dependent upon its local environment, this providesunequivocal definition of its partitioning into phospholipid membranes.In aqueous conditions, tryptophan typically emits light near 355 nm whenexcited with 285 nm light. In contrast, the emitted light is red-shiftedor lowered when the tryptophan of the peptide is present in an apolarenvironment, such as the interior of a phospholipid membrane, andproduces a maximum emission wavelength typically in the range of 325 to345 nm. Both the magnitude and wavelength of light emitted under thisfluorescence assay are influenced by the properties of the peptide andthe composition of the phospholipid membrane. For example, negativelycharged groups present in the membrane may reduce the intensity of theemitted signal and weakly interacting peptides may undergo only a slightdecrease in maximum emission wavelength. A typical assay employed an 8μM solution of promolecules and liposomes of defined size (100 nm indiameter, 0.5 mM) composed of phosphatidylserine or phosphatidylcholineor a combination thereof to test the influence of negatively charged orzwitterionic surfaces. Alternatively, detergent micelles of sodiumdodecylsulphate (10 mM) or dodecylphosphocholine (5 mM) were used inplace of liposomes, as they were easier to prepare and provided similarresults. Time-dependent measurement of the fluorescence emission spectrawas used to characterize the activation of the promolecules by thrombin(2 nM) or by other proteases (10 nM or higher).

Analysis by Förster Resonance Enemy Transfer

Förster resonance energy transfer (FRET) relies upon the ability oflight to be exchanged from one fluorescent moiety to another, whichbecomes possible only when the moieties are in close proximity to oneanother, typically less than about 30 Angstroms. Liposomes havingsimilar composition to that used in the intrinsic fluorescence assayswere prepared with 1% DiO, a lipid-soluble fluorescent compound whosefluorescence emission characteristics match the excitation wavelength ofthe promolecule when conjugated to Cy5. DiO-containing liposomes (1 μM)were mixed with Cy5 containing promolecules or activated peptideportions (20 nM), and the spectral characteristics of the mixture weredetermined. Cy3 was excited with 545 nm light, and the emission of Cy5was measured over 650 to 700 nm. The activated peptide portions, but notthe promolecules, enabled fluorescence energy transfer, which confirmedthe observations made using intrinsic fluorescence.

Analysis by Circular Dichroism Spectroscopy

Circular dichroism (CD) spectroscopy relies upon the features of theentire peptide and, in particular, its ability to adopt a more compactand regular structure that differentially absorbs polarized light. Whena molecule adopts an alpha-helical structure, a stronger differentialsignal results, particularly over the range of 215 to 400 nanometers.All CD spectra were recorded using a JASCO J710 spectropolarimeter at25° C., with a cell of 1 mm path length. The CD spectra were obtainedusing a measurement range from 260 to 190 nm, a 1 nm bandwidth, and 100nm/min scanning speed. CD spectra of peptides in a concentration rangingfrom 0.02 to 0.1 mM in phosphate buffered saline in the presence orabsence of SDS (20 mM) were obtained. Similar to the results obtainedwith techniques using fluorescence, the activated peptide portions, butnot the promolecules, underwent a more prominent conformational changein the presence of sodium dodecylsulphate micelles.

Characterization of Interaction with Eukarvotic Cells in Suspension

Interaction of the promolecule and its activated peptide form witheukaryotic cell membranes was performed using Jurkat cells thatnaturally grow in suspension (approximately 100,000 cells in a volume of100 μL) by incubating the cells with peptides for 2 hours at 37° C.Dulbecco's modified Eagle medium containing 1% fetal bovine serum wasused as the cell growth medium. Cell viability was assessed using Trypanblue dye exclusion. Limited toxicity was observed even under highconcentrations of the promolecule.

To assess selective activation, the assays were repeated with theexogenous addition of thrombin (5 nM), plasmin (200 nM), and coagulationfactor Xa (200 nM). Despite their presence in 40-fold higher abundance,neither plasmin nor factor Xa activated the promolecules (50 μM) withsufficient efficiency to mediate cell death based on trypan blue dyeexclusion. In contrast, thrombin efficiently converted the promoleculesinto the activated peptide form, which mediated cell death in a mannersimilar to that observed by direct addition of the activated form of thepromolecule.

Characterization of Interaction with Eukarvotic Cells on Plasticware

To examine the interaction of the promolecule and its activated formwith cell membranes rather than artificial membranes, the promoleculeswere incubated with cells of the breast cancer cell line MDA-MB-231(approximately 100,000 cells in a volume of 100 μL) for 1 hour at 37° C.in Dulbecco's modified Eagle medium containing 1% fetal bovine serum,and cell viability was assessed using Trypan blue dye exclusion. Limitedtoxicity was observed even under high concentrations of the promolecule.

Analysis Using DRAQ7 Exclusion

DRAQ7 is a far-red fluorescent DNA dye that stains the nuclei of deadand permeabilized cells. Unlike trypan blue, it can be combined withother commonly used fluorophores, such as Oregon Green 488 and Cy5.Healthy cells exclude the dye, and no increase in fluorescence isobserved. Dead or damaged cells do not exclude the dye, and the dyeenters the cells, binds to DNA, and becomes fluorescent. When complexedwith double stranded DNA, the dye has a maximum excitation wavelength of643 nm and a maximum emission wavelength of 694 nm. Cells incubated withthe promolecule or the activated peptide form of the promolecule in thepresence or absence of thrombin (5 nM) were exposed to DRAQ7 (3 μM) forat least 10 minutes and then imaged using two different approaches. Inthe first approach, the cells were mouse pancreatic duct adenocarcinomacells, the incubation buffer contained wheat germ agglutinin conjugatedwith Oregon Green 488, and the cells were visualized with a NikonEclipse Ti-E bearing the appropriate filters. In the second approach,mouse X3 fibroblast cells bearing a modified Histone 2A gene fused togreen fluorescent protein (GFP) were imaged using an Acumen Ex3—acommercially available confocal microscopy instrument specificallydesigned for high throughput, content rich and highly multiplexed dataapplicable to a wide range of biological assays.

Analysis of Blood Clots Formed on Glass Slides

Whole bovine blood was mixed at a ratio of 5:1 with a 10 nM solution ofpromolecules bearing the fluorescent dye Cy5. The mixture was then addedto 1 uL of activated coagulation factor Xa (10 nM) and calcium chloride(10 mM) to initiate clot formation on the surface of a conventionalmicroscopy slide. The mixture was then stirred with the tip of a plasticpipette for four minutes until a clot was formed. The clot was thenpulled gently across the slide with the goal of flattening out the clotfor better imaging. A coverslip was then placed on top of the slide andheld in place by dabbing the corners with adhesive. The slide was thenimaged within a few hours using an epifluorescence microscope (NikonEclipse Ti-E) bearing the appropriate filters for detection of the Cy5dye.

Analysis in Animal Models

Fox N1 nude mice were used for imaging studies when the dye ATTO 680 wasconjugated to the promolecule. Unlike Cy5, the ATTO 680 dye enablesdetection using longer wavelengths that have less auto-fluorescence fromthe animal, and is therefore more suitable for imaging studies usinganimal models. Preliminary estimates indicated that the probe bearingthe imaging agent was cleared rapidly, having a circulating half-life often minutes or less. This short circulating half-life is extremelybeneficial for imaging, as the background signal dissipates rapidly. Twodifferent models of in vivo clot formation were investigated. In bothmodels, a solution of the promolecule (0.5 nmol) was injected into thetail vein or inferior vena cava of the mouse as a first step in theprocedure.

In the first model, thromboplastin was subsequently administereddirectly into the inferior vena cava at two different doses. The firstdose was fatal to mice within a few minutes of administration. Thesecond dose was five-fold lower than the first dose, and was not fatalto mice within the time frame of the experiment. The thromboplastinmodel was performed according to Weiss E J, Hamilton J R, Lease K E,Coughlin S R. (2002) Protection against thrombosis in mice 15 lackingPAR3, Blood 100(9):3240-4. This approach induces systemic blood clotformation and results in blockage of thinner vasculature in the lungs,and aims to be more reflective of the pulmonary emboli present in aclinical population. The promolecule clearly identified lungs bearing ahigher burden of clots despite the blocked vasculature and hence limitedability of the promolecule to accumulate over time.

In the second model, a 28-gauge needle was used to wound the animal inthe hind leg, and the progress of clot formation was measured over time.In this approach, the clot formed is generated over a period of time andis more substantial in magnitude. Clot formation was readily detectedwithin minutes, and the intensity of the signal reached a plateau withinthirty minutes.

Example 1 Promolecules Containing Temporin-L for the Detection of BloodClots

Three or four polypeptide modules were combined to form variouspromolecules capable of controllable cell membrane interaction. Aschematic diagram of a promolecule containing four polypeptide modulesis shown in FIG. 1. The naturally-occurring sequence of Temporin-L(FVQWFSKFLGRIL (SEQ ID NO: 2)) was modified to restrict non-specificactivation by other trypsin-like proteases. The modified sequence(FVQWFSKFLGKLL (SEQ ID NO: 3)) was used as portion A of the promolecule.In order to form the cleavable linker X², the amino acid sequence PR wasadded to the C-terminus of portion A.

The naturally-occurring thrombin substrate PAR-1 (SFLLRNPNDKYEPFW (SEQID NO: 18)) was modified to reduce its positive charge and add morenegative charge. The modifications resulted in the amino acid sequenceSFLLQDPNDQYEPFW (SEQ ID NO: 19), which was used to form portion Z of thepromolecule. The amino acid sequence of the promolecule containingportions A, X², and Z, but not optional portion X^(1a) or X^(1b), wastherefore: FVQWFSKFLGKLLPRSFLLQDPNDQYEPFW (SEQ ID NO: 47). Promoleculescontaining optional portion X^(1a) were also formed. These promoleculeswere formed by adding the amino acid cysteine to the N-terminus ofportion A and then conjugating an imaging agent to the cysteine residue.Imaging agents used included Cy5 fluorescent dye and ATTO 680fluorescent dye.

Once all versions of the promolecule had been synthesized, they werecharacterized in a number of ways. Intrinsic fluorescence analysis, FRETanalysts, and circular dichroism spectroscopy, as described above, wereused to analyze the inactive promolecule and the active form of thepromolecule. In the presence of phospholipid liposomes or detergentmicelles, the active form of the promolecule demonstrated a decrease inmaximum emission wavelength using intrinsic tryptophan fluorescence(FIG. 5). The activate form, but not the inactive promolecule form, wascapable of FRET to appropriately fluorescent liposomes when the peptidewas conjugated to the Cy5 fluorescent dye (FIG. 6). The activate formalso demonstrated an increase in alpha-helical content via circulardichroism spectroscopy (FIG. 7). In contrast, the inactive promoleculeform showed no binding to liposomes and limited interaction withdetergents, indicating that activation had successfully changed thecharacteristics and properties of the promolecule.

Selective activation of the promolecule by thrombin was confirmed bothin vitro and in vivo by incubating the promolecule with eukaryotic cellsand examining trypan blue and/or DRAQ7 dye exclusion, as describedabove, in the presence or absence of exogenous proteases and theirinhibitors. At nanomolar concentrations, thrombin activated thepromolecule, while several other proteases did not (FIG. 8). Even atmuch higher concentrations, other proteases did not activate thepromolecule, illustrating the high selectivity of the PAR-1 sequenceused for protease recognition (FIG. 9). Blocking the activity ofthrombin with the known inhibitor hirudin confirmed that thrombinmediated the observed results.

Toxicity was determined by incubating eukaryotic cells with variousconcentrations of the promolecule or the activated peptide form andanalyzing trypan blue and/or DRAQ7 dye exclusion, as described above.After prolonged incubation at peptide-to-cell ratios greater than 10⁹ toone, which occur at high micromolar concentrations of the promoleculethat are well above the intended diagnostic range, the incubated cellsdid not exclude trypan blue dye or DRAQ7 fluorescent dye, indicatingthat the cell membranes had been destabilized and the cells were nolonger viable (FIGS. 10 and 11). Selective activation by thrombin wasobserved using a DRAQ7-based assay, described above, wherein 5 nM ofthrombin and 100 μM of the promolecule were incubated with the cells forup to 60 minutes. The results showed that the cells gradually lost theability to exclude the DRAQ7 dye over the time frame of the experiment,indicating that the promolecule was activated by thrombin and associatedwith the cell membranes (FIG. 12).

Analysis of blood clots formed on glass slides, as described above, wasused to test localization of the promolecule to sites of thrombinactivity. Briefly, clots were formed on glass slides with or withoutinitiation by coagulation factor Xa and calcium chloride in the presenceof a promolecule containing Cy5 conjugated via the thiol of the cysteineamino acid added to the N terminus of portion A. Actively clottingplatelets were clearly associated with intense accumulation of theactivated peptide form of the promolecule (FIG. 13). Without activation,the promolecule did not accumulate on red blood cells. These resultsindicate that the promolecule could be activated by initiated bloodclots, and that the activated form of the promolecule could thenlocalize to the site of an initiated blood clot.

To test activity in vivo, promolecules were injected into mice using twomodels of thrombosis, described above. In the first model, 500 picomolesof a promolecule wherein X¹ was ATTO 680 conjugated via a cysteineresidue was injected into the inferior vena cava of a mouse.Subsequently, a fatal or non-fatal dose of thromboplastin wasadministered to the mouse. A fatal dose was defined as a dose thatresulted in animal death within 20 minutes of injection. Followingadministration of the fatal or non-fatal dose of thromboplastin, thelungs of the animal were surgically removed, visually inspected, andimaged using fluorescence microscopy (FIG. 14). By visual inspection,emboli and damaged lung tissue were evident based on a white appearanceof the lungs. Analysis of the fluorescence signal resulting from theATTO 680 showed that the activated from of the promolecule accumulatedat sites of emboli deposition in the lungs.

In a second in vivo model, a 28-gauge needle was used to create a smallwound in the hind leg of nude mice after injection of 500 picomoles ofthe same promolecule described above. The resulting wound was nearlyinvisible to the naked eye, yet readily accumulated the fluorescentsignal within minutes (FIG. 15). Signal intensity was monitored inreal-time, producing a progress curve that plateaued within twentyminutes (FIG. 16). These results demonstrated that the promolecule iscapable of imaging blood clots in vivo that are less than 1 mm³ with ahigh signal-to-noise ratio as well as quantifying the magnitude of theirformation. Using a standard curve of the fluorescent signal, the localconcentration was calculated to be 0.5 μM, or 10¹² molecules, of theactivated form of the promolecule in the blood clot that was formed. Inthe absence of wounding, the activated form of the promoleculeaccumulated in the duodenum at sites of digestion and was clearedrapidly (FIG. 17).

Example 2 Promolecules Containing Protonectin for the Detection of BloodClots

Protonectin is an antimicrobial peptide that is found in the venom ofthe neotropical wasp Agelaia pallipes. The naturally-occurring sequenceof protonectin (ILGTILGLLKGL (SEQ ID NO: 4)) was used to form portion Aof a promolecule. Portion X² was formed from amino acid residues PR, andthe modified PAR-1 exosite recognition sequence as described in Example1 (SFLLQDPNDQYEPFW (SEQ ID NO: 19)) was used to form portion Z. A lysineresidue was added to the N-terminus of portion A, and Cy3 fluorescentdye was conjugated to the lysine residue. The combination of thesemodules resulted in a promolecule having the sequence(Cy3-K)ILGTILGLLKGLPRSFLLRNPNDKYEPFW (SEQ ID NO: 48).

The promolecule was digested with 0.1 nanomoles of thrombin, followed byanalysis with reverse phase HPLC to verify digestion (FIG. 18).Digestion was approximated by the Michaelis-Menten kinetic parametersk_(cat)/K_(M)=8 μM⁻¹ sec⁻¹ and K_(M) 200 nM (data/calculations notshown). Portion A of this promolecule lacked a tryptophan residue, andtherefore it could not be analyzed using intrinsic fluorescence. FRETanalysis, as described above, revealed that membrane-interactingpeptides comprising protonectin interact with negatively chargedphospholipid membranes, such as phosphatidylserine (FIG. 19). Blood clotformation on glass slides was used to test localization of the activatedform of the promolecule to sites of thrombin activity. Blood clots wereformed on glass slides with or without initiation by coagulation factorXa and calcium chloride in the presence of the promolecule. Activelyclotting platelets were clearly associated with intense accumulation ofthe active form of the promolecule as evidenced by visualization of Cy3via fluorescence microscopy (FIG. 20). Without activation, thepromolecule did not accumulate on red blood cells. These resultsindicated that a promolecule containing protonectin as portion A couldbe activated by initiated blood clots, and that the active form of thepromolecule could then localize to the site of an initiated blood clot.

Example 3 Promolecules Containing Temporin-L for the Detection ofMatriptase Activity in Cancer

Many proteases have been suggested as potential biomarkers or drugtargets for breast cancer, yet they remain underutilized because of thedifficulty of detecting proteolysis in vivo. Restricted interactionpeptides could therefore be used to diagnose such cancers in addition tobeing useful as reagents to understand the biology underlying theseprocesses (FIG. 21). Examples of proteases involved with breast cancerinclude prostate specific antigen, cathepsin B, stromelysin 3, matrixmetalloproteinase 2, urokinase-type plasminogen activator, andmatriptase, among others.

A promolecule was created whose X² linker could be cleaved bymatriptase, a multidomain membrane-bound serine protease also known asMTSP-1, commonly over-expressed in infiltrating breast carcinomas. Themodified Temporin-L peptide sequence described in Example 1(FVQWFSKFLGKLL (SEQ ID NO: 3)) was used to form portion A, and thematriptase cleavage sequence KSR was used to form cleavable linker X². Apeptide sequence derived from the naturally-occurring substrate vascularendothelial growth factor receptor 2 (VEGFR2) with three additionalnegative charges to lower the overall pI of the promolecule was used toform portion Z (AEDEGLYDDD (SEQ ID NO: 21)). The combination of thesemodules resulted in a promolecule having the sequence:

(SEQ ID NO: 49) (Cy5)FVQWFSKFLGKLLKSRAEDEGLYDDD.

Intrinsic fluorescence analysis, as described above, showed that thepromolecule formed from these three portions (A-X²—Z) exhibited moderaterestricted interaction with phospholipid membranes and exhibited lowtoxicity towards MDA-MB-231 cells (FIGS. 22 and 23). Exogenenousaddition of different proteases showed that this promolecule wasselectively activated by matriptase, as indicated by diminished trypanblue dye exclusion (FIG. 24). Cy5 fluorescent dye was conjugated to thispromolecule via an N-terminal thiol (module X^(1a)) to create apromolecule having the structure X^(1a)-A-X²-Z, and its interaction withHT29 cells was analyzed using fluorescence microscopy. In thisexperiment, cells were grown to confluence on glass bottom microwelldishes and the peptide (10 uM) incubated with the cells for between oneto three hours and then washed for staining with fluorescently-labeledwheat germ agglutinin. After such staining the cells were covered intypical cell culture media and allowed to grow overnight for anotherround of imaging. Results showed that the activated form of thepromolecule accumulated on the surface of the HT29 cells, which areknown to express matriptase. Washing the cells to remove unboundmolecules, followed by further incubation, revealed that the activatedform of the promolecule was internalized into the cells with the naturalprocess of membrane recycling (FIG. 25).

Example 4 Promolecules Containing Temporin-L for the Detection ofTMPRSS2 Activity in Cancer

A promolecule is synthesized having a portion A sequence ofFVQWFSKFLGKLL (SEQ ID NO: 3) derived from Temporin-L, a cleavable linkerportion X² having the sequence RSLIG (SEQ ID NO: 91), which, whencontiguously linked to the modified Temporin-L sequence of portion Aforms the sequence LLRSLIG (SEQ ID NO: 30), which is recognized andcleaved by the enzyme TMPRSS2, which is known to be associated withmetastatic cancer, a portion Z sequence of KVDGTSHVTGDDD (SEQ ID NO:20), which is known as a protease activated receptor-2 (PAR-2) thatspecifically interacts with the TMPRSS2 enzyme, and Cy5 fluorescent dyeas the detectable moiety on portion X^(1a). The combination of thesemodules results in a promolecule having the sequence:

(SEQ ID NO: 50) (Cy5)FVQWFSKFLGKLLRSLIGKVDGTSHVTGDDD.

This promolecule is administered to a subject suspected of havingprostate cancer. Once administered to the subject, the promolecule comesinto contact with TMPRSS2 enzymes located on the apical membranes ofprostate epithelial cells. Upon contacting the TMPRSS2 enzymes, portionis X² is cleaved, causing the cleavage product containing portion A toseparate from portion Z and undergo a conformational change, forming analpha-helical structure and inserting into cell membranes in thevicinity of the cleavage. Subsequently, prostate epithelial cells areimaged using fluorescence microscopy to detect the Cy5 fluorescent dye.Detection of the Cy5 dye facilitates diagnosis of prostate cancer in thesubject.

Example 5 Promolecules Containing Temporin-L for the Detection of theSecreted Aspartyl Proteases of Pathogenic Fungi

A promolecule is synthesized having a portion A sequence ofFVQWFSKFLGKLL (SEQ ID NO: 3) derived from Temporin-L, a cleavable linkerportion X² having the sequence VELLYLV (SEQ ID NO: 31) (cleaved by theenzyme Secreted Aspartyl Peptidase (SAP)), a portion Z sequence of DD,and Cy5 fluorescent dye as the detectable moiety on portion X^(1a). Thecombination of these modules results in a promolecule having thesequence: (Cy5)FVQWFSKFLGKLLVELLYLVDD (SEQ ID NO: 51).

This promolecule may be administered to a subject and used to detect thepresence of pathogens that utilize extracellular proteases as virulencefactors. Secreted aspartyl peptidases (SAPs), which are known to besecreted by Aspergillus and Candida fungi, cleave the X² linker havingthe sequence VELLYLV (SEQ ID NO: 31), causing the cleavage productcontaining portion A to undergo a conformational change, forming analpha-helical structure and inserting into cell membranes in thevicinity of the cleavage. Subsequently, areas of the subject suspectedof being infected with pathogenic organisms are imaged usingfluorescence microscopy to detect the Cy5 fluorescent dye. Detection ofthe Cy5 dye facilitates diagnosis of an infection.

Example 6 Design of a Compound for Increased Circulating Half-Life

A promolecule having the same structure as that described above inExample 1, with a cysteine residue attached to the N-terminus of portionA and a molecule of Cy5 fluorescent dye conjugated through portionX^(1a) is synthesized (the overall structure beingCy5-CFVQWFSKFLGKLLPRSFLLQDPNDQYEPFW (SEQ ID NO: 52)). In order to extendthe circulating half-life of the promolecule, it is covalently linked toa molecule of polyethylene glycol (PEG) having a moleculer weight of1,000 Daltons. The PEG-modified promolecule is administered to a subjectand the circulating half-life is measured. The PEG-modified form of thepromolecule has a longer circulating half-life than the non-PEG-modifiedform of the promolecule.

Example 7 Promolecule Requiring One of Two Possible Events forActivation

A promolecule having the overall structure X^(1a)-A-X^(2a)-X^(2b)-Z issynthesized, wherein portion A has the sequence FFWLSKIF (SEQ ID NO:11), portion Z has the sequence DD, and portion X^(1a) comprises acysteine residue, which is attached to the N-terminus of portion A andconjugated to a molecule of Cy5 fluorescent dye. Portion X^(2a) has thesequence PR, which is cleaved by the enzyme thrombin. Portion X^(2b) hasthe sequence DDDDK (SEQ ID NO: 41), which is cleaved by the enzymeenteropeptidase. The combination of these modules results in apromolecule having the sequence:

(SEQ ID NO: 53) (Cy5)CFFWLSKIFPRDDDDKDD.

In order to activate this promolecule, either X^(2a) or X^(2b) must becleaved, so that this promolecule is activated by either thrombin orenteropeptidase activity. The molecule is administered to a subject, andis cleaved in areas having enteropeptidase or thrombin activity, causingportion A to separate from portion Z and interact with membranes in thevicinity. Areas of a subject suspected of having enteropeptidase orthrombin activity are examined using fluorescence microscopy, and theCy5 dye is detected in areas having enteropeptidase or thrombinactivity.

Example 8 Design of a Promolecule Requiring Two Eevents for Activation

A promolecule having the overall structure:

is synthesized, wherein portion A has the sequence FFWLSKIF (SEQ ID NO:11), portion Z has the sequence DD, and portion X^(1a) comprises acysteine residue attached to the N-terminus of portion A conjugated to amolecule of Cy5 fluorescent dye. Portion X^(2a) has the sequence PR,which is cleaved by the enzyme thrombin. Portion X^(2b) has the sequenceDDDDK (SEQ ID NO: 41), which is cleaved by the enzyme enteropeptidase.

In order to activate this promolecule, both X^(2a) and X^(2b) must becleaved, so that this promolecule is activated in areas where boththrombin and enteropeptidase are expressed. The molecule is administeredto a subject, and is cleaved in areas having both enteropeptidase andthrombin activity, causing portion A to separate from portion Z andinteract with membranes in the vicinity. Areas of the subject suspectedof having enteropeptidase and thrombin activity are examined usingfluorescence microscopy, and Cy5 dye is detected in areas having bothenteropeptidase and thrombin activity.

Example 9 Promolecules Containing Temporin-SHf for the Detection ofBlood Clots Via the Activity of Coagulation Factor IXa

A promolecule is synthesized having a portion A sequence of *FFWLSKIF(SEQ ID NO: 11) (which is derived from Temporin-SHf), a cleavable linkerportion X² having the sequence NGRI (SEQ ID NO: 46) and a portion Zhaving the sequence HNQS(X)SDD, wherein X is either Y, H or Q (SEQ IDNO: 22), which are cleavage sequences of the coagulation factor IXa, anda portion X^(1a) whose N-terminus is derivatized with the fluorescentmoiety fluorescein. The combination of these modules results in apromolecule having the sequence: *FEWLSKIENGRIHNQS(X)SDD, wherein X iseither Y, H or Q (SEQ ID NO: 54). * indicates the fluorescent moietyfluorescein.

This promolecule is administered to a subject suspected of having activeclotting involving coagulation factor IXa. Once administered to thesubject, the promolecule comes into contact with coagulation factor IXain active clots. Upon contacting the coagulation factor IXa, portion X²is cleaved, causing the cleavage product containing portion A to undergoa conformational change, forming an alpha-helical structure andinserting into cell membranes in the vicinity of the cleavage.Subsequently, areas of the subject suspected of having clotting activityinvolving coagulation factor IXa are imaged using fluorescencemicroscopy to detect the fluorescein dye. Detection of fluoresceinfacilitates diagnosis of active clotting in the subject.

Example 10 Design of Promolecules Bearing a Different Order of Modules

A promolecule is synthesized having the following formula:X^(1b)-Z-X²-A. Amino acid sequences of promolecules having this generalformula are listed below in Table 5 (below).

TABLE 5 Promolecules having a different order of peptide modules.*indicates the presence of a detectable moiety at the N-terminus, theC-terminus, or both of the peptide. Target Protease Z-X²-A SEQ ID NO:Thrombin *-DDD-LVPRS-FFWLSKIF SEQ ID NO: 55 aPC *-DDD-LVKRS-FFWLSKIF SEQID NO: 56 Intermediate 1 *-DDD-LVGRS-FFWLSKIF SEQ ID NO: 57 Factor Xa*-DDD-LEGRS-FFWLSKIF SEQ ID NO: 58 Intermediate 2 *-DDD-LVVRS-FFWLSKIFSEQ ID NO: 59 Factor IXa *-DDD-LVVRL-FFWLSKIF SEQ ID NO: 60 ThrombinDDD-LVPRS-FFWLSKIF-* SEQ ID NO: 55 aPC DDD-LVKRS-FFWLSKIF-* SEQ ID NO:56 Intermediate 1 DDD-LVGRS-FFWLSKIF-* SEQ ID NO: 57 Factor XaDDD-LEGRS-FFWLSKIF-* SEQ ID NO: 58 Intermediate 2 DDD-LVVRS-FFWLSKIF-*SEQ ID NO: 59 Factor IXa DDD-LVVRL-FFWLSKIF-* SEQ ID NO: 60 Thrombin*-DDD-LVPRS-FFWLSKIF-* SEQ ID NO: 55 aPC *-DDD-LVKRS-FFWLSKIF-* SEQ IDNO: 56 Intermediate 1 *-DDD-LVGRS-FFWLSKIF-* SEQ ID NO: 57 Factor Xa*-DDD-LEGRS-FFWLSKIF-* SEQ ID NO: 58 Intermediate 2*-DDD-LVVRS-FFWLSKIF-* SEQ ID NO: 59 Factor IXa *-DDD-LVVRL-FFWLSKIF-*SEQ ID NO: 60

This promolecule is administered to a subject suspected of having activeclotting involving any of the target proteases listed in Table 5. Onceadministered to the subject, the promolecule comes into contact with thetarget protease in active clots. Upon contacting the target protease,portion X² is cleaved, causing the cleavage product containing portion Ato undergo a conformational change, forming an alpha-helical structureand inserting into cell membranes in the vicinity of the cleavage.Subsequently, areas of the subject suspected of having clotting activityinvolving the target protease are imaged to detect the detectablemoiety. Detection of the detectable moiety facilitates diagnosis ofactive clotting in the subject.

While the present invention has been described with reference to thespecific embodiments thereof, it should be understood by those skilledin the art that various changes may be made and equivalents may besubstituted without departing from the true spirit and scope of theinvention. In addition, many modifications may be made to adapt aparticular situation, material, composition of matter, process, processstep or steps, to the objective, spirit and scope of the presentinvention. All such modifications are intended to be within the scope ofthe claims appended hereto.

What is claimed is:
 1. A molecule comprising the structure, fromN-terminal to C-terminal,X^(1a)-A-X²-Z-X^(1b) wherein: X^(1a) and/or X^(1b) may be present orabsent, and when present comprise a nucleophilic moiety; A is amembrane-interacting polypeptide portion comprising the amino acidsequence FVQWFSKFLGKLL (SEQ ID NO: 3); X² is a cleavable linker which iscleavable by thrombin, wherein X² joins portion A to portion Z; and Zcomprises an exosite recognition sequence for thrombin.
 2. The moleculeof claim 1, wherein portion Z comprises a covalently linked watersoluble polymer.
 3. The molecule of claim 1, wherein the exositerecognition sequence for thrombin comprises the thrombin exositerecognition sequence of a protease-activated receptor-1 (PAR-1).
 4. Themolecule of claim 3, wherein Z comprises the amino acid sequenceSFLL(X^(a))NPNDKYEPFW wherein X^(a) is R or Q (SEQ ID NO: 23).
 5. Themolecule of claim 1, wherein one or more of X^(1a), X^(1b), A, or Zcomprises a D-amino acid.
 6. The molecule of claim 1, wherein X^(1a) ispresent and comprises a nucleophilic moiety.
 7. The molecule of claim 1,wherein X^(1b) is present and comprises a nucleophilic moiety.
 8. Themolecule of claim 6 or claim 7, wherein the nucleophilic moiety ofX^(1a) or X^(1b) comprises a thiol functional group.
 9. The molecule ofclaim 6 or claim 7, wherein X^(1a) or X^(1b) comprises an amino acidresidue comprising the nucleophilic moiety.
 10. The molecule of claim 9,wherein the amino acid residue is a cysteine residue.
 11. The moleculeof claim 9, wherein the amino acid residue is a lysine residue.
 12. Themolecule of claim 6 or claim 7, wherein X^(1a) or X^(1b) comprises acargo moiety covalently attached to the nucleophilic moiety.
 13. Themolecule of claim 12, wherein the cargo moiety is a detectable moiety.14. The molecule of claim 13, wherein the detectable moiety comprises afluorescent moiety.
 15. The molecule of claim 13, wherein the detectablemoiety comprises a radioisotope.
 16. A composition comprising: themolecule of claim 1; and a pharmaceutically acceptable carrier.
 17. Amethod of detectably labeling a cell, the method comprising: contactinga cell with the molecule of claim 13; wherein when said contacting isunder conditions suitable for cleavage of the cleavable linker, themolecule is cleaved to release the membrane interacting polypeptideportion for interaction with a phospholipid bilayer of the cell anddetectably labels the cell.
 18. The method of claim 17, wherein the cellis in vivo.
 19. A method for detection of a blood clot in a subject, themethod comprising: administering to the subject a molecule of claim 13,wherein, in the presence of thrombin, the molecule is cleaved to releasea cleavage product comprising the detectable moiety and the membraneinteracting polypeptide portion and wherein the cleavage productinteracts with a phospholipid bilayer of a cell in an area of thrombinenzyme activity; and detecting the presence or absence of the detectablelabel of the cleavage product; wherein the presence of the detectablelabel indicates an area of thrombin enzyme activity associated withactive clotting.
 20. The method of claim 19, wherein the exositerecognition sequence for thrombin comprises the exosite recognitionsequence of protease-activated receptor-1 (PAR-1) (SEQ ID NO: 18). 21.The method of claim 19, wherein the subject is a human.
 22. A method ofmaking a molecule useful in delivery of a cargo moiety to a phospholipidbilayer, the method comprising: synthesizing the molecule of claim 1,wherein X^(1a) is present; and attaching a cargo moiety to thenucleophilic moiety of X^(1a); wherein a molecule useful in delivery ofa cargo moiety to a phospholipid bilayer is produced.
 23. The method ofclaim 22, wherein said synthesizing is by chemical synthesis.
 24. Themolecule of claim 1, wherein Z comprises the amino acid sequenceSFLLQDPNDQYEPFW (SEQ ID NO: 19).
 25. The molecule of claim 1, whereinA-X²-Z comprises the amino acid sequence FVQWFSKFLGKLLPRSFLLQDPNDQYEPFW(SEQ ID NO: 47).
 26. The molecule of claim 13, wherein Z comprises theamino acid sequenceSFLL(X^(a))NPNDKYEPFW (SEQ ID NO: 23) wherein X^(a) is R or Q.
 27. Themolecule of claim 13, wherein Z comprises the amino acid sequenceSFLLQDPNDQYEPFW (SEQ ID NO: 19).
 28. The molecule of claim 13, whereinA-X²-Z comprises the amino acid sequence FVQWFSKFLGKLLPRSFLLQDPNDQYEPFW(SEQ ID NO: 47).
 29. The composition of claim 16, wherein Z comprisesthe amino acid sequenceSFLL(X^(a))NPNDKYEPFW (SEQ ID NO: 23) wherein X^(a) is R or Q.
 30. Thecomposition of claim 16, wherein Z comprises the amino acid sequenceSFLLQDPNDQYEPFW (SEQ ID NO: 19).
 31. The composition of claim 16,wherein A-X²-Z comprises the amino acid sequenceFVQWFSKFLGKLLPRSFLLQDPNDQYEPFW (SEQ ID NO: 47).
 32. The composition ofclaim 31, wherein X^(1a) or X^(1b) comprises a nucleophilic moietycovalently attached to a detectable moiety.
 33. The method of claim 19,wherein Z comprises the amino acid sequenceSFLL(X^(a))NPNDKYEPFW (SEQ ID NO: 23) wherein X^(a) is R or Q.
 34. Themethod of claim 19, wherein Z comprises the amino acid sequenceSFLLQDPNDQYEPFW (SEQ ID NO: 19).
 35. The method of claim 19, whereinA-X²-Z comprises the amino acid sequence FVQWFSKFLGKLLPRSFLLQDPNDQYEPFW(SEQ ID NO: 47).